Table of Contents
ISAC XXIII International Congress
May 20 – 24, 2006
Quebec City Convention Center
Quebec City, Canada
ISAC Leadership and Congress Organizers ............................................................................................................... 6
Program Schedule .......................................................................................................................................................... 7-12
Quebec City Convention Center Maps ............................................................................................................... 13-15
General Information ................................................................................................................................................... 16-17
Registration and Service Desk
Business Center
CMLE Accreditation
Coat Check/Luggage Storage
Congress Evaluation
Cyber Café
Emerging Leaders Club
Exhibits Location and Hours
Oral Sessions/Speaker Practice
Poster Sessions
Social Events
Scientific Program
Keynote Lecture ............................................................................................................................................................ 18
Robert Hooke Distinguished Lecture ..................................................................................................................... 19
Scientific Tutorials .................................................................................................................................................. 20-25
Frontiers lecture Series ......................................................................................................................................... 26-30
Plenary Sessions ..................................................................................................................................................... 31-38
Parallel Sessions ..................................................................................................................................................... 39-45
New Investigator/Student Symposium ................................................................................................................. 45
Outstanding Poster Award Applicants ........................................................................................................... 46-47
Poster Presentations ............................................................................................................................................. 48-62
Workshops ................................................................................................................................................................ 63-67
Commercial Tutorials .................................................................................................................................................. 68-75
Exhibit Hall Floor Plan ................................................................................................................................................ 76-77
Exhibitor List .................................................................................................................................................................. 78-88
Abstracts ....................................................................................................................................................................... 89-225
Abstract Author Index ........................................................................................................................................... 226-233
Congress Sponsors ................................................................................................................................................. 235-237
ISAC Membership Application ................................................................................................................................... 239
ISAC 2006 Program and Abstracts
3
Save the Date!
The International Society for Analytical Cytology
is proud to announce the
ISAC XXIV International Congress
17 - 21 May 2008
Budapest, Hungary
ISAC XXIII
INTERNATIONAL CONGRESS
Dear Colleagues,
Welcome to the XXIII Congress of the International Society for Analytical Cytology. Québec City
is a beautiful city in which to enjoy the environment as well as take the opportunity to enhance
your scientific insights in cytometry.
This year our meeting opens up a new dimension. The field of Cytomics has become more
mature and depends upon the unique capabilities of cellular analysis systems to thrive. ISAC is
the leading society focused on quantitative analysis of the cell. We utilize a variety of technologies
at the DNA, RNA, and protein levels, and integrative approaches to elucidate the relationships
between molecules, molecular complexes, and networks responsible for function and behavior
of complex systems. Development and application of novel quantitative molecular approaches
to measure the properties of single cells and complex tissues is at the heart of ISAC’s raison
d’être. This year’s Congress expands our role and highlights new opportunities in the world of
biological imaging. ISAC brings knowledge and experience in using quantitative approaches to
deal with large data sets of single-cell measurements. New tools to elucidate information on
interacting molecules and pathways and models to predict behavior and function requiring a
broad spectrum of analytical tools are being presented at this Congress.
The scientific program has been changed significantly for this Congress, with emphasis on
bringing the entire Congress together for more sessions as a whole. Thus we have initiated a
frontiers and a plenary session for the whole Congress each day. Two highlights will be Roger
Tsien’s Keynote address on Saturday and Stephen Lewis’ Hooke lecture on Sunday evening. All
the posters will be available for viewing during the entire Congress. Poster sessions are vital
interactive components of a Congress and I encourage you to spend time reviewing them.
The scientific program is organized around three themes – biological sciences, clinical sciences,
and cytometric technology – and includes (in addition to the frontiers and plenary sessions) a
daily parallel session, as well as workshops on Monday, Tuesday, and Wednesday. The awards
ceremony will be on Wednesday evening, followed by the final-night reception and banquet.
You may have noted that we have two pre-Congress courses which are being directed by
members of ISAC’s emerging leaders. These short courses complement the Congress by offering
opportunities for members to learn different analytical approaches.
Our exhibitors, many of whom are new to the ISAC Congress, play an integral part in the success
of our Congress. This year we attracted a record number of exhibitors and a record amount of
sponsorship. Please make sure you spend time in the exhibit hall to visit with exhibitors and see
what they have to offer.
The ISAC XXIII Congress promises to be an exciting and memorable scientific meeting. I would
personally value your feedback after you’ve reflected on the week with your friends and
colleagues.
J. Paul Robinson
President-Elect, Congress Chair
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ISAC Leadership
ISAC Executive Committee
Congress Organizing Committee
President
Maria G. Pallavicini
Secretary
Robert M. Zucker
Congress Chair
Congress Co-chair
President-Elect
J. Paul Robinson
Treasurer
Frank N. Traganos
J. Paul Robinson
Alex Nakeff
Marcel Desrosiers
Gary Durack
Mike Keeney
Lori Krueger
Phil Marder
Robert Murphy
John Nolan
Robert Zucker
Yuval Garini
Gerald Gregori
Lara Krebs
Bartek Rajwa
Directors, Flow and
Imaging Pre-Congress
Courses
Laura Teodori
Zofia Maciorowski
Membership Services
(Travel and Awards)
Past President
Harry A. Crissman
Councilors
Julie Auger
Jan W. Gratama
Lori Krueger
Robert F. Murphy
Alexander Nakeff
John P. Nolan
David R. Parks
Paul Smith
János Szöllosi
áá
Laura Teodori
Headquarters Staff
Executive Director
Richard Koepke
FASEB Congress Management
Geri Swindle
Jean Lash
Crystal Hiner
About the International Society for Analytical Cytology
ISAC is a scientific and educational organization whose purposes are:
To promote research, development, and applications in analytical cytology. Analytical cytology is
broadly defined as the characterization and measurement of cells and cellular constituents for
biological, diagnostic and therapeutic purposes. It embraces components of cytochemistry, cytophysics,
anatomy, biology, physiology, pathology, image analysis, instrumentation, clinical laboratory practice
and other subjects of relevance.
To facilitate integration of the many disciplines within analytical cytology.
To disseminate knowledge of analytical cytology.
To provide information and advice on those aspects of public policy which are concerned with
analytical cytology.
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Program Schedule
11:30 – 13:00
Saturday, 20 May
08:00 – 09:30
SCIENTIFIC TUTORIAL SESSIONS
Tutorial 1: Slide-Based
Room 202
Cytometry
Atilla Tarnok
Tutorial 2: Image Analysis
Of Subcellular Patterns for
High Throughput Screening
and Systems Biology
Robert Murphy
Room 301
Tutorial 3: Flow Cytometry
of Bacteria and Yeast
Susann Müller
Room 302
Tutorial 4: Circulating
Endothelial Cells and
Endothelial Progenitor Cells
Phil McCoy
Room 303
Tutorial 5: Fluorescent Probe Room 304
Technology
Alan Waggoner
09:45 – 11:15
Tutorial 6: Live Cell Imaging Room 202
Simon Watkins
Tutorial 9: The Diagnosis of Room 303
Myelodysplastic Syndromes by
Flow Cytometry
Brent Wood
Tutorial 10: Polychromatic Room 304
Flow Cytometry
William Telford and Steve Perfetto
Room 202
Tutorial 12: Particle and
Organelle Tracking
Gaudenz Danuser
Room 301
Tutorial 13: FRET for
Monitoring Molecular
Associations: A Practical
Approach
János Szöllosi
Room 302
Tutorial 14: Antigen-Specific Room 303
Cytometry
Andreas Thiel and Mario Assenmacher
Tutorial 15: Cytometry of
Room 304
Cell Cycle and Apoptosis
Zbigniew Darzynkiewicz and Frank Traganos
13:00 – 14:30
Lunch
14:30 – 14:45
Welcome and Opening Remarks
14:45 – 15:45
Tutorial 7: Basics of Machine Room 301
Learning for Image or Flow
Robert Murphy
Tutorial 8: Intracellular
Room 302
Signaling and Phosphoprotein
Analysis
Omar Perez
Tutorial 11: Confocal
Microscope Quality
Assurance
Robert Zucker
FRONTIERS 1
Chair: John Nolan
Single Cell Kinase Signaling
for Mechanistic and Clinical
Analyses
Garry Nolan
Room 200C
15:45 – 16:45
Location Proteomics: Image
Informatics for Systems
Biology
Robert Murphy
Room 200C
16:45 - 17:45
KEYNOTE LECTURE
Chair: J. Paul Robinson
Molecules Crafted to Spy
on Cells and Tumors
Roger Tsien
Room 200C
18:00 – 19:00
Opening Reception
Room 200A
7
13:00 – 14:00
Sunday, 21 May
08:00 – 09:30
PARALLEL SESSION 1
Advanced Microscopy and
Image Acquisition 1
Room 301
Image Processing and
Analysis 1
Room 302
Flow Instrumentation 1
Room 303
Cell Physiology 1
Room 304
Calibration and
Standardization
Room 202
09:30 – 10:00
Break
Exhibit Hall
10:00 – 11:00
FRONTIERS 2
Chair: Phillip Marder
NIH Roadmap Initiative
Overview
Jim Inglese
Room 200C
11:00 – 12:00
12:00 – 16:00
12:00 – 13:00
12:00 – 13:00
13:00 – 14:00
13:00 – 14:00
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High Throughput Flow
Cytometry and the NIH
Roadmap Molecular
Libraries Initiative
Larry Sklar
Room 200C
Exhibits Open
Exhibit Hall
400 B/C
COMMERCIAL TUTORIALS
New Lasers. New Optics.
Room 301
New BD FACSDiva™
Software. Innovations and
Developments at BD Biosciences
BD Biosciences
Applications for Large
Particle Flow Cytometry
Union Biometrica, Inc.
14:00 – 14:30
PLENARY SESSION 1
Chair: Robert Murphy
Multimode Live Cell Imaging Room 200C
Reveals a Novel Method of
Cellular Communication in
the Immune System
Simon Watkins
14:30 – 15:00
Fluorescent Speckle
Microscopy
Gaudenz Danuser
Room 200C
15:00 – 15:30
Facile Generation of
Room 200C
Biosensors to Study
Endogenous Protein Activation
in Living Cells
Klaus Hahn
15:30 – 16:00
Break
16:00 – 17:30
WORKSHOPS
1. Cell Based Imaging
Using High Content
Screening and Analysis
Joe Trask
Exhibit Hall
Room 202
2. Hot Topics in Apoptosis
Andrea Cossarizza
205 B/C
3. Biosafety
Ingrid Schmid
Room 301
4. Flow Cytometric
Room 302
Analysis of Leukemia and
Lymphoma
Mike Borowitz and Brent Wood
Room 204A
Flow Cytometric Analysis of Room 302
Signal Transduction Networks
Beckman Coulter, Inc.
Accessing Dynamic
Parameters Within Cells by
Combining Imaging and
Fluorescence Fluctuation
Evotec Technologies
Use of Qdot® Nanocrystals for Room 202
Imaging and Flow Cytometry
Applications
Invitrogen
Room 303
5. Flow Technology
Instrument Developments
John Nolan
Room 303
6. Cytometry Education
Lori Krueger
Room 304
17:45 – 18:45
19:00 – 21:00
ROBERT HOOKE
Room 200C
DISTINGUISHED
LECTURE
Chair: Maria Pallavicini
Science and Advocacy: The Best
Hope to Defeat the Pandemic
of AIDS
Stephen Lewis
Congress Exhibitor Mixer/ Exhibit
Hall
Exhibits Open/Poster Viewing
12:00 – 13:00
Standards Rule! A Program
Room 204A
for Standardized Instrument
QC, Setup and Quantitative
Fluorescence Measurements
Bangs Laboratories, Inc.
12:00 – 13:00
Cytometer Set-up and
Tracking in a Multi-color,
Digital World
BD Biosciences
Room 301
12:00 – 13:00
High Content Imaging:
Taking Cell Analysis to a
Higher Resolution
BD Biosciences
Room 302
12:00 – 13:00
Maximizing Use of the
Violet Laser
Invitrogen
Room 202
12:00 – 13:00
FlowJo Basics 101: Learn
Mo’ About the ‘Jo
Tree Star, Inc.
Room 304
12:30 – 13:30
Use of Activation State
Specific Antibodies in Flow
and Imaging Cytometric
Applications
Cell Signaling Technology
Room 303
13:00 – 14:00
Cell Cycle Analysis in
Live Cells
Invitrogen
Room 202
13:00 – 14:00
Software Solution for
Bead-Based Immunoassays
Soft Flow Hungary Ltd.
Room 204B
14:00 – 17:30
HCDM Workshop
Room 304
Monday, 22 May
08:00 – 9:30
PARALLEL SESSION 2
Advanced Microscopy
and Image Acquisition 2
Room 301
Analysis 2
Room 302
Room 303
Cell Physiology 2
Room 304
Rare Event Detection and
Stem Cell Technologies
Room 202
09:30 – 10:00
Break
Exhibit Hall
09:30 – 14:00
Exhibits Open
Exhibit Hall
400 B/C
10:00– 11:00
11:00 – 12:00
12:00 – 14:00
FRONTIERS 3
Chair: Paul Smith
Protein Engineering by
Yeast Surface Display and
Flow Cytometric Sorting
Dane Wittrup
Room 200C
The Pharmacodynamics of
Room 200C
Molecular Cancer Therapeutics
David Hedley
COMMERICAL TUTORIALS
Every Dot Has a Story:
205 B/C
Multispectral Image
Analysis of Cells in Flow with
the Amnis Imagestream
System
Amnis Corporation
14:00 – 14:30
14:30 – 15:00
PLENARY SESSION 2
Chair: Michael Keeney
Multiplexed Hybrid
Room 200C
Cytometry-Based Application
Platform Technology: A Tool to
Fight the HIV Pandemic in
the 21st Century in HIV
Frank Mandy
Regulation and Therapeutic Room 200C
Targeting of Human Leukemia
Stem Cells
Kristen Hope
9
15:00 – 15:30
Diagnosing PNH and
Room 200C
Previously Undiagnosed
Acute Leukemias with FLAER
and Multiparameter Flow
Cytometry
Robert Sutherland
15:30 – 16:00
Break
Exhibit Hall
15:30 – 19:30
Exhibits Open
Exhibit Hall
400 B/C
16:00 – 17:00
Awards Competition
Chairs: Laura Teodori and
Zofia Maciorowski
Room 200C
17:00 – 18:00
ISAC Business Meeting
Room 200C
18:00 – 19:30
Poster Presentations 1
and Happy Hour
Exhibit Hall
Tuesday, 23 May
08:00 – 9:30
PARALLEL SESSION 3
Analysis 3
Room 301
BioPharma Applications
Room 302
Room 303
Cell Physiology 3
Room 304
Immune Monitoring
Room 202
09:30 – 10:00
Break
Exhibit Hall
09:30 – 14:00
Exhibits Open
Exhibit Hall
400 B/C
10:00 – 11:00
11:00 – 12:00
10
FRONTIERS 4
Chair: Lori Krueger
Searching for an HIV
Vaccine - The Role of
Flow Cytometry
Mario Roederer
Human Stem Cell Biology
Limitations and Potential
Applications
Mickie Bhatia
12:00 – 13:00
COMMERCIAL TUTORIALS
Lyoplate Technology: A
New Paradigm for FlowBased Assay Design and
Implementation
BD Biosciences
Room 301
12:00 – 13:00
cGMP Sorting of
Hematopoietic Stem Cells
Dako
12:00 – 13:00
FlowJo Advanced: New
Room 304
Features and Advanced Topics
Tree Star, Inc.
13:00 – 14:00
TBD
13:00 – 14:00
A System for Automated
Room 303
Isolation of Single Cell Clones
Evotec Technologies
13:00 – 14:00
Routine Detection and
Room 204A
Quantitation of Fetomaternal
Hemorrhage Using Fetal
Cell Count™
IQ Products
13:00 – 14:00
Dyes, Assays, and Workflow Room 202
Solutions for High Throughput
Imaging (Hcs/Hti)
Invitrogen
13:00 – 14:00
PhycoLink® Conjugation
and Purification Kits: How
to Make the Best Darn
Conjugates
Prozyme, Inc.
14:00 – 14:30
Room 200C
PLENARY SESSION 3
Chair: Alexander Nakeff
High Content Analysis in
Drug Discovery
Joe Trask
Room 302
Room 204B
Room
205 B/C
Room 200C
14:30 – 15:00
Rare Event Detection in
Solid Cancers
Alison Allan
Room 200C
15:00 – 15:30
Image-Based Screen for
Cell Cycle and Cancer
Targets
Dan Rines
Room 200C
Room 200C
15:30 – 19:30
Exhibits Open
Exhibit Hall
400 B/C
Wednesday, 24 May
15:30 – 16:00
Break
Exhibit Hall
08:00 – 9:30
16:00 – 17:30
WORKSHOPS
7. Cytometric
Approaches for Biomarker
Research
Phil Marder
PARALLEL SESSION 4
Image Spectral Analysis
Room 301
Biotechnology
Room 302
Apoptosis
Room 303
Cancer Biomarkers
Room 304
Environmental, Marine,
and Microbiology
Room 202
09:30 – 10:00
Break
Exhibit Hall
09:30 – 12:00
Exhibits Open
Exhibit Hall
400 B/C
10:00 – 11:00
FRONTIERS 5
Fulwyler Session
Chemical Cytometry
Analytical Chemistry of
Single Cells
Norm Dovichi
Room 202
8. Image Segmentation
Jelena Kovacevic
Room 301
9. Multiplexed and
Microparticle Assays
Marie Iannone
Room 302
10. Cell Functional
Analysis
George Babcock and
Padma Narayanan
Room 303
11. Spectroscopy
Issues and Cytometry
Jeremy Lerner, Steven
Lockett and Robert Zucker
Room 304
12. Cytometry Applications
in Biotechnology
Gary Elliott
Room
205 B/C
18:00 – 19:30
Poster Presentations 2
and Happy Hour
Exhibit Hall
400 B/C
19:00 – 22:30
Resource Manager’s
Workshop
Room 204A
11:00 – 12:00
14:00 – 14:30
The LCOS-Based
Programmable Array
Microscope (PAM)
Tom Jovin
PLENARY SESSION 4
Chair: Robert Zucker
A Human Protein Atlas
for Normal and Cancer
Tissues
Mathias Uhlen
Room 200C
Room 200C
Room 200C
14:30 – 15:00
Flow and Image Cytometric
FRET for Detecting Protein
Associations
János Szöllosi
Room 200C
15:00 – 15:30
Do Not Mind The Gap:
Protein Trafficking between
the Endoplasmic Reticulum
and the Golgi Apparatus in
Plant Cells
Federica Brandizzi
Room 200C
15:30 – 16:00
Break
11
16:00 – 17:30
WORKSHOPS
13. Tracking
Room 202
Microorganisms at the Single
Cell Level: Industrial,
Environment and Aquatic
Microbiology
Michel Denis
14. Astrobiology
Sarah Baatout
Room 301
15. Topics in Multi-color
Room 302
Immunoflorescence:
Appropriate Use of Isotype
Controls
Lori Krueger and Ruud Hulspas
12
16. Flow Cytometric
Analysis of ZAP-70
in B-CLL: Are There
Alternatives?
Jan Phillipe
Room 303
17. Laser Scanning
Cytometry
Attila Tarnock
Room 304
18. Flow Cytometry
Calibration and
Standardization
Robert Zucker
Room
205 B/C
17:30 – 18:45
Break
18:45 – 20:00
Awards Ceremony
Room 200C
20:00 – 23:00
Congress Banquet
Room
200 A/B
Convention Center Layout - Level 2
13
14
15
General Information
All activities for the Congress are located in the Québec City
Convention Center except the Introductory Courses scheduled
at Laval University.
Congress Evaluation
Registration and Service Desk – Foyer 2, Level 4
After the Congress you will receive an online evaluation form.
Please complete the evaluation of the Congress.
The ISAC Congress Organizing Committee needs your input to
help make the ISAC XXIV International Congress even better.
Registration will take place at the ISAC registration desk at the
following times:
Cyber Café – Exhibit Hall, Level 4
Friday, 19 May
Saturday, 20 May
Sunday, 21 May
Monday, 22 May
Tuesday, 23 May
Wednesday, 24 May
15:00
07:30
07:30
07:30
07:30
07:30
–
–
–
–
–
–
18:00
18:00
18:00
17:00
17:00
13:00
Badges/Access Control
Participation in the ISAC Congress is limited to registered
attendees. The official badge is required for admittance to all
sessions, social activities and the exhibit hall. A fee may be
charged to reissue lost or misplaced badges. Please do not
place a business card into the badge holder as identification. If
there is an error on your badge, please have it corrected at the
registration desk.
Companions/Guest Registration
Companions and spouses of registered attendees are welcome
to attend ISAC’s Congress. They may register in the ISAC
registration area located in Foyer 2. Registered companions
and guests are invited to attend the opening reception and
final night banquet. The companion registration fee is $100.
Business Center – Level 3
There is a Business Center/Concierge in the Convention Center.
Services include fax, phone, secretarial services office supplies,
souvenirs and reservations.
CMLE Accreditation
This continuing medical laboratory education activity is
recognized by the American Society of Clinical Pathologists
(ASCP) as meeting the criteria for 44 hours of credit. ASCP
CMLE credit hours are acceptable to meet the continuing
education requirement for the ASCP Board of Registry
Continuing Competence Recognition Program. CMLE materials
are available at the registration desk.
Coat Check/Luggage Storage – Main Lobby,
Level 4
Facilities for luggage storage and coat check will be available
Saturday – Wednesday. Please do not bring luggage to the
meeting rooms.
16
The Cyber Café will be open during exhibit and poster hours.
Emerging Leaders Club – Solarium, Level 3
Recognizing that it is important to reach out and bring together
young and first-time registrants, ISAC established the“Emerging
Leaders Club”. Visit the Club and mix and mingle with your
peers from many diverse disciplines and countries around the
world. The Club will be open Saturday through Wednesday.
Exhibits – Exhibit Hall 400B/C, Level 4
The Congress will be highlighted by an Exposition featuring
displays by leading suppliers and vendors. A complete directory
and floor plan of exhibiting companies is included in the
program. Visit the exhibits during the following hours:
Sunday, 21 May
12:00 – 16:00
19:00 – 21:00
Exhibits Open
Exhibits Open/Mixer
Monday, 22 May
09:30 – 14:00
15:30 – 19:30
Exhibits Open
Exhibits Open
Tuesday, 23 May
09:30 – 14:00
15:30 – 19:30
Exhibits Open
Exhibits Open
Wednesday, May 24
09:30 – 12:00
Exhibits Open
Oral Sessions/Speaker Practice – Room 201B,
Level 2
It is important that all speakers go to the speaker practice room
to review and check the compatibility of your presentation
with the AV equipment at least four hours prior to speaking. An
audiovisual technician is available in the room to assist speakers.
Poster Sessions – Exhibit Hall, 400 B/C, Level 4
Tours
More than 350 posters will be presented.The posters are divided
into two sessions to allow attendees to spend time with the
presenters.
Québec City is truly unique. The only walled city north of
Mexico, it is proud of a history blending French and English
influences – from the stone walls that encircle the old city and
the Citadelle keeping watch over the St. Lawrence to the
Martello towers and the Parliament Building, where Québec
politics have been played out for over a century. These ever
present witnesses to times past are now fascinating places to
visit and explore, and played a major role in the city’s
designation as a UNESCO world heritage treasure. Not far from
Old Québec, at Domaine Maizerets the Maison des Jésuites,
the Trait-Carré and the Maison Hamel-Bruneau, rich treasures
of art and architecture and other vestiges of the past also attest
to the life and lifestyle of bygone days.
For information on things to do and see during your visit go to
www.quebecregion.com.
Posters must be placed on the assigned poster board by 09:30
on Sunday, 21 May. Posters are to remain on the board through
12:00 Wednesday, 24 May. Poster materials should be removed
promptly at 12:00 on Wednesday. Posters will be available for
viewing Sunday through Tuesday from 09:30 – 19:30 and on
Wednesday from 09:30 – 12:00. Poster authors will be at their
poster for presentation and discussion as follows:
Monday, 22 May
Odd-numbered poster boards – 18:00 -19:30
Tuesday, 23 May
Even-numbered poster boards – 18:00 -19:30
The poster presentations are numbered 149/P1 to 489/P365.
The first three digit number is the program/abstract number.
The P1-P365 is the poster board number.
SOCIAL EVENTS
Congress Opening Reception ......................................... Room 200A
Saturday, 20 May/18:00 – 19:00
All registered participants are invited to the Congress Opening
Reception immediately following the Keynote Lecture.
Congress Exhibitor Mixer ................................................. Exhibit Hall
Sunday, 21 May/19:00 – 21:00
All registered participants are invited to attend the Congress
Exhibitor Mixer Sunday evening.
Meet the exhibitors, network with your colleagues and enjoy
the evening with friends.
Congress Gala Dinner ....................................................... Room 200A
Wednesday 24 May/20:00 – 23:00
The Congress will close with a dinner dance so bring your
dancing shoes! Tickets will be required for the dinner. An
exchange coupon is included with your registration badge.
The coupon must be exchanged for a ticket before 17:00
Monday, 21 May.
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Keynote Lecture
1. Molecules Crafted to Spy on
Cells and Tumors .......................... Room 200C
Chair: J. Paul Robinson
Roger Tsi en
Howard Hughes Medical
Institute, UCSD
Genetically encoded tags and indicators are molecular spies
that reveal specific gene products and biochemical processes
in living cells and organisms. Florescent proteins from jellyfish
and corals have been bred to eliminate multimerization and
cover the entire visible spectrum. Somatic hyper mutation in B
lymphoma cells harnesses the immune system to produce a
powerful new way to evolve protein properties. Indicators
constructed from fluorescent proteins can report local dynamic
signals such as redox potential, neurotransmitter
concentrations, protein-protein interactions, and kinase vs.
phosphatase activities.
Although fluorescent proteins are powerful tools, they cannot
be reduced below ~220 amino acids, and their only useful
readout is fluorescence. Much shorter peptide sequences
combine genetic encoding with the greater range of
spectroscopic properties available by organic synthesis.
Tetracysteine motifs of 6-12 amino acids can be labeled in live
cells with membrane-permeant biarsenical dyes. Unique
applications include green vs. red pulse-chase labeling of old
vs. new copies of the same protein, electron-microscopic
localization, chromophore-assisted light inactivation of a chosen
protein without the problems of antibody penetration, and
measurement of local Ca2+ within nanometers of proteins such
as Ca2+ channels.
For clinical applications one would prefer not to have to
introduce genes or be limited to optical detection. Argininerich sequences are known to mediate uptake of a wide variety
of contrast agents into cells and tissues in vivo. We find that
such uptake can be prevented by appending certain polyanionic
sequences and selectively re-activated by cleavage of the linker.
This new mechanism offers the exciting possibility that
radioactive, magnetic, and infrared contrast agents and
therapeutic drugs may be concentrated in diseased tissues
expressing particular extra cellular proteases.
18
Saturday, 20 May
16:45 – 17:45
Roger Y. Tsien received his A.B. in Chemistry and Physics
summa cum laude from Harvard College in 1972. A Marshall
Scholarship then took him to the Physiological Laboratory at
the University of Cambridge, where he received his Ph.D. in
1977 and remained as a Research Fellow until 1981. He then
became an Assistant, Associate, then full Professor in the Dept.
of Physiology-Anatomy at the University of California, Berkeley.
In 1989 he moved to the University of California, San Diego,
where he is an Investigator at the Howard Hughes Medical
Institute and Professor in the Depts. of Pharmacology and of
Chemistry & Biochemistry. In 1996 he was a scientific cofounder of Aurora Biosciences Corporation, which went public
in 1997 and was acquired by Vertex Pharmaceuticals in 2001.
In 1999 he was a scientific co-founder of Senomyx, Inc.
Dr. Tsien’s research has been at the interfaces between organic
chemistry, cell biology, and neurobiology, starting long before
such interdisciplinary efforts became fashionable. He is best
known for designing and building molecules that either report
or perturb signal transduction inside living cells.These molecules,
created by organic synthesis or by engineering naturally
fluorescent proteins, have enabled many laboratories including
his to gain new insights into signaling via calcium, sodium, pH,
cyclic nucleotides, nitric oxide, inositol polyphosphates,
membrane potential changes, protein phosphorylation, active
export of proteins from the nucleus, and gene transcription.
The optical reporter molecules are also valuable in miniaturized
high-throughput screening of candidate drugs in the
pharmaceutical industry. His current research goals are to
understand how the spatial and temporal dynamics of signal
transduction orchestrate complex cellular responses such as
gene expression and synaptic plasticity.These goals will require
improved molecular techniques to see and manipulate smallmolecule messengers, protein phosphorylation, and proteinprotein interaction in live cells and organisms. He is also
developing new ways to target contrast agents and therapeutic
agents to tumor cells based on their expression of extra cellular
proteases.
Robert Hooke
Distinguished Lecture
Science and Advocacy:
The Best Hope to Defeat the
Pandemic of AIDS ........................ Room 200C
Stephen Lewis
UN Secretary General’s
Special Envoy for HIV/
AIDS in Africa
Sunday, 21 May
17:45 – 18:45
From 1984 through 1988, Stephen Lewis was the Canadian
Ambassador to the United Nations. In this capacity, he chaired
the Committee that drafted the Five-Year UN Program on
African Economic Recovery. He also chaired the first
International Conference on Climate Change, which drew up
the first comprehensive policy on global warming.
Mr. Lewis holds 22 honorary degrees from Canadian universities
and is an honorary fellow of the Royal College of Physicians
and Surgeons of Canada. In May 2003, in recognition of
outstanding contributions to public health, Columbia
University’s Mailman School of Public Health honored him with
the Dean’s Distinguished Service Award.
Mr. Lewis was appointed a Companion of the Order of Canada,
Canada’s highest honor for lifetime achievement, in 2003. The
same year, Maclean’s magazine honored Mr. Lewis as their
inaugural “Canadian of the Year.”
Stephen Lewis is the UN Secretary-General’s Special Envoy for
HIV/AIDS in Africa, a post he’s held since June 2001. He is also
a Commissioner for the World Health Organization’s
Commission on the Social Determinants of Health, and a Senior
Advisor to the Mailman School of Public Health at Columbia
University in New York. Mr. Lewis is also a director of the Stephen
Lewis Foundation, which is dedicated to easing the pain of
HIV/AIDS in Africa.
His work with the UN has shaped the past two decades of his
career. From 1995 to 1999, Mr. Lewis was Deputy Executive
Director of UNICEF at the organization’s global headquarters in
New York.
In March 2004, Mr. Lewis was honored by the United Nations
Association in Canada with the Pearson Peace Medal, which
celebrates outstanding achievements in the field of
international service and understanding.
In April 2005, TIME magazine listed Stephen Lewis as one of
the “100 most influential people in the world.” The same year,
the International Council of Nurses awarded Mr. Lewis their
prestigious Health and Human Rights Award, which is awarded
quadrennially for outstanding contributions to international
health and human rights.
Stephen Lewis’ book, Race Against Time was released in October
2005, and is published by House of Anansi Press.
In 1997, in addition to his work at UNICEF, he was appointed by
the Organization of African Unity to a Panel of Eminent
Personalities to Investigate the Genocide in Rwanda. The
“Rwanda Report” was issued in June of 2000. In 1993, Mr. Lewis
became coordinator for the international study — known as
the Graça Machel study — on the “Consequences of Armed
Conflict on Children”. The report was tabled in the United
Nations in 1995.
19
Scientific Tutorials
Saturday, 20 May
08:00 – 09:30
An additional registration fee is required to participate in the scientific tutorials program. If you have not
pre-registered for tutorials you may register on-site at the registration desk as long as space is available.
Session I
Tutorial 1: Slide-Based Cytometry ...... Room 202
Atilla Tarnok
Level: Intermediate to advanced (basic cytometry and
microscopy knowledge in techniques, methods, assays, biology
required)
Expected audience: Core facility and lab managers, physicians
(oncology, immunology, pathology), PhD students (probably
some technicians)
therefore be appropriate for those interested in using existing
software for these tasks but also for those interested in
developing or improving such software. Basic knowledge of
fluorescence microscopy and digital image acquisition will be
expected. The tutorial is anticipated to be especially useful for
those running core cytometry facilities who would like to
expand the level of automated image analysis available to their
users.
Tutorial 3: Flow Cytometry of
Bacteria and Yeast ................................ Room 302
Topics covered: Basics of Slide-Based Cytometry (concept,
instrumentation, sample and data handling, providers); General
Applications (immunology, cell biology, histology, clinicals
examples); Specific Applications (e.g. poly- and hyperchromatic
analysis, cytomics and tissomics)
Tutorial 2: Image Analysis of Sub-cellular
Patterns for High Throughput Screening
and Systems Biology ........................... Room 301
Robert Murphy
High throughput microscopy is well-established for cell-based
drug screening and will play a growing role in systems biology
efforts by providing fundamental information on the subcellular
distributions of proteins and other biological macromolecules.
This tutorial will describe methods for automated analysis of
subcellular patterns in fluorescence microscope images,
applicable to all types of traditional and high throughput
microscopy (wide-field, deconvolution, confocal, structuredlight, multi-photon). Topics covered will be:
• Recommendations regarding image acquisition for
subsequent automated analysis;
• Methods for automated segmentation of multi-cell images
into single cell regions;
• Description of types of features used to describe
subcellular patterns;
• Methods for extraction of these features (especially
morphological, texture and wavelet features);
• Statistical and machine learning methods for comparison,
classification and clustering of patterns;
• Publicly available image database systems.
The tutorial will cover concepts and algorithms but also give
practical examples of how these tasks are accomplished. It will
20
Susann Müller
Microorganisms are seen as catalytic systems which are
employed both in biotechnology and biodegradation of toxic
compounds in the environment. The physiological states of
the individuals within the populations are heterogeneous,
despite of growing them in pure or mixed cultures and all the
more when investigating natural populations. Microbial
performances strongly depend on the state of the individual in
the cell cycle and on its specific strategy to meet changing
(micro)environmental conditions. As a result, the individual
metabolic performances are disparate.
Microbial Multiparametric Cytometry will be shown to be able
to follow individual physiological characteristics of bacteria as
well as yeast cells by employing several structural and functional
parameters. Examples will be shown where subpopulations
were fluorescently differentiated and spatially separated by
cell sorting and further investigated by involving genetic tools
and proteomics. The individual strategies of microorganisms,
enabling them for the man-wished performances, will be
envisioned.The application of these techniques for optimizing
industrial processes in biotechnology by tuning the
microenvironmental conditions to the needs of the
microorganism will also be presented.
Saturday, 20 May
08:00 – 09:30
Tutorial 4: Circulating Endothelial Cells and
Endothelial Progenitor Cells ............... Room 303
Tutorial 5: Fluorescent Probe
Technology ........................................... Room 304
J. Phillip McCoy, Jr.
Alan Waggoner
The endothelium, which comprises the lining of blood vessels,
is one of the largest and most complex organs in the human
body. Blood vessel development and repair is a complex process
involving the migration, adherence, and proliferation of
circulating endothelial cells (CECs) either from pre-existing
blood vessels (angiogenesis) or from the differentiation of
endothelial progenitor cells (EPCs) (vasculogenesis). Recent
studies have provided evidence that cells capable of both
revascularization and neovascularization are present in the
peripheral circulation in extremely low frequencies. The study
of endothelial cells and their precursors is challenging. Not
only are there no markers that have exacting specificity for
these cells, but also the maturation from a precursor to a mature
endothelial cell appears to be a continuum rather than occurring
through discrete stages.
This tutorial will cover basics of fluorescence needed to
understand the use of fluorescent probes in flow cytometry
and fluorescence imaging. It will cover photo-physics, the
excited state of fluorescent dyes, fluorescent labels, fluorescent
physiological indicators, energy transfer, fluorescence
polarization, two-photon imaging, fluorescence detection
components and instrumentation and signal-to-noise issues. It
is intended for intermediate level scientists with basic chemistry,
physics and biology courses and modest experience in
cytometry.
Circulating CECs and EPCs have been reported as biomarkers
of both angiogenesis and of vascular damage in various
diseases. Thus there is an increasing demand for assays capable
of providing accurate and reproducible identification and
enumeration of these cells in the peripheral circulation.
This tutorial will review methods for the detection of circulating
endothelial cells (CECs) and endothelial progenitor cells (EPCs).
Flow cytometric approaches for identifying these extremely
rare cells will be presented and data will be presented validating
these methods.
21
Saturday, 20 May
09:45 - 11:15
Session II
Tutorial 6: Live Cell Imaging ................ Room 202
Simon Watkins
Returning science to the living cell, tissue or organisms is the
goal of post-genomic research. Microscopic imaging tools are
one of the principal methodologies which may be applied to
the living system. In fact developments in detectors, computing,
and fluorescent proteins have moved these approaches to the
center of basic research in living systems.
This tutorial concentrates on live cell imaging using
fluorescence methods. The presentation is focused on how to
optimize the entire microscope system for live cell imaging,
including optimization of the stand, automation, objectives and
detectors. Fluorescent proteins and their use and comparative
value will also be presented as well as discussions of the merits
of newer methods such as TIRF and multiphoton imaging,
Tutorial 7: Basics of MachineLearning for
Image or Flow ................................................. Room 301
Robert Murphy
Machine learning methods are playing an increasing role in
both image and flow cytometry. This intermediate-level tutorial
will describe the basic concepts and paradigms of machine
learning (the “3C’s” of machine learning: Comparison,
Classification, Clustering) and provide practical information on
applying them to cytometry data. Topics covered will include:
• The multivariate data matrix and its descriptive statistics.
• Comparison: Are two samples the same?
o Parametric methods
o Non-parametric methods (including tree-based
methods)
o Influence of sample size
• Classification: Which of a set of known classes should a
new sample be assigned to?
o Linear Discriminant Analysis
o Classification Trees
o Neural Networks
o Support Vector Machines
o Ensemble Classifiers
o Bayesian Classifiers
• Clustering: What classes are present in a sample?
o Basic clustering methods
o Methods for determining number of clusters
o Consensus clustering methods
o Methods for comparing clusterings
o Co-clustering
• Machine Learning tools for Cytometry
22
The tutorial will NOT cover the processes for collecting
cytometry data or extracting descriptors/features. It WILL
assume at least basic exposure to algorithms and statistics.
The tutorial is expected to be useful for basic and clinical
cytometry researchers interested in applying cutting-edge
machine learning methods to their data.
Tutorial 8: Intracellular Signaling and
Phosphoprotein Analysis .................... Room 302
Omar Perez
Functional outcomes of cellular processes are typically
mediated by the integration of intracellular signal transduction
pathways. Multidimensional flow cytometry of intracellular
protein phospho-epitopes allows the study multiple signaling
processes simultaneously. This approach allows correlation of
intracellular protein activation with surface
immunophenotypes, cell cycle status, apoptosis, or additional
parameters. Correlations of these internal cellular states are
important in immune cell function and may be relevant to
disease manifestation of oncologies or autoimmune disease.
This tutorial is intended for both beginners and advanced multicolor experts. Many of the examples come from problems in
immunology, and will describe approaches involving a single
parameter and working up to 12-parameter multi-color
protocols.
Tutorial 9: The Diagnosis of Myelodysplastic
Syndromes by Flow Cytometry .......... Room 303
Brent Wood
The diagnosis of myelodysplastic syndromes in many cases
relies on nonspecific clinical findings and subjective assessment
of peripheral blood and bone marrow morphologic changes.
Flow cytometric abnormalities have been described in these
disorders and offer the potential for more accurate and
objective diagnosis and classification.
This tutorial will review the patterns of maturation seen in
normal bone marrow, illustrate the types of abnormalities
commonly identified in patients with myelodysplastic
syndromes and myeloproliferative disorders, and discuss their
potential clinical utility.
Saturday, 20 May
08:00 – 09:30
Tutorial 10: Polychromatic Flow
Cytometry ............................................ Room 304
Steve Perfetto and William Telford
This tutorial will describe the objectives, procedures, analysis
and instrumentation needed to perform successful
polychromatic flow cytometry (PFC). Subjects will include (1)
an overview of the fluorescent probes available for multicolor
flow, with particular attention to novel fluorochromes such as
quantum nanoparticles, (2) appropriate fluorochrome selection
in multicolor systems, (3) monoclonal antibody reagent titration,
(3) instrument alignment and calibration, (4) specimen controls,
(5) instrument configuration and (6) multiparametric data
analysis, including compensation. Real-world polychromatic
labeling data (from six to twenty colors) will be used to illustrate
the issues involved in designing and analyzing a useful
polychromatic labeling scheme. Both commercially available
instrumentation and new trends in instrument design (including
novel laser sources) will also be discussed as platforms for
adding additional parameters to existing multicolor labeling
systems. Special attention will be paid to color compensation,
including choosing the right controls, using software-based
compensation systems, and selecting the right fluorochromes
to minimize compensation problems.
23
Saturday, 20 May
11:30 - 13:00
Session III
Tutorial 11: Confocal Microscope
Quality Assurance ................................ Room 202
Robert Zucker
The goal of many CLSM experiments is to quantify
fluorescence. The accuracy of these measurements requires
that the system be properly aligned with correct spectral
registration. In many laboratories, the most common method
to check the performance of a CLSM system is to characterize
a histological slide to create a “pretty picture”. Unfortunately
this approach is inadequate for our cytometry field. A series of
tests have been developed to replace this subjective approach;
these include the following: field illumination, spectral
registration, colocalization, lens function, total laser power, laser
stability, dichroic reflectance, axial resolution, galvanometer
scanning stability, stage drift, overall machine stability, and
system noise. Improvements and modifications of these tests
will be described in addition to new methods in the detection
of system instability. Recently a new spectral performance test
has been developed using the MIDL lamp to measure the
spectral accuracy of all confocal spectral imaging systems.This
test will help investigators assess both the performance of their
instruments as well as the quality of their spectral data.
This tutorial assumes that the participant will have a basic
knowledge of the confocal microscope operation. It will focus
on the Quality Assurance aspects of this technology. The tutorial
will be extremely beneficial for all scientists that utilize this
technology in their research.
Tutorial 12: Particle and
Organelle Tracking ............................... Room 301
This tutorial will cover four aspects of building such computer
vision systems for live cell imaging:
• Detection of sub-cellular structures by adaptive statistical
methods;
• Methods for the assignment of corresponding structures
in consecutive frames;
• Classification of particle motion in the case of diffusion,
super-diffusion, convective flows; and of particle motion
dictated by higher order regulatory schemes;
• Tracking of sub-cellular structures with low image stability,
i.e. the methods discussed under aspect 1 fail to provide
particle coordinates that can be robustly assigned over
multiple frames.
The tutorial will explain these steps based on a number of cell
biological applications:
• Cell-based screening of metastasis-blocking agents;
• Quantitative genetics of the regulation of chromosome
dynamics in yeast;
• Quantitative genetics of axonal transport in Drosophila
larvae;
• Cargo dependent endocytosis;
• Actin and microtubule cytoskeleton dynamics in cell
migration and mitosis;
• Molecular coupling in cell adhesion structures.
We will cover some mathematical principles, but the tutorial
will be kept on a conceptual level that can be appreciated by a
mathematically less experienced audience.
Tutorial 13: FRET for Monitoring
Molecular Associations:
Following the dynamics of sub-cellular structures is increasingly A Practical Approach ........................... Room 302
Gaudenz Danuser
becoming the method of choice to study molecular
mechanisms in all cell functions and is rapidly gaining attraction
as a readout of cell behavior in screens of the genome,
proteome, or small molecule libraries. These applications all
rely on the ability to track the complex motion of subcellular
structures and to classify their behavior in the framework of
distinctive mathematical parameters or mechanistic models
of the underlying molecular processes.This requires a complete
tracking of all structures, including statistically rare events, which
is no longer achievable by manual or semi-automatic means.
Instead, computer vision methods have to be developed that
guarantee fully automatic and reliable tracking without user
intervention.
24
János Szöllõsi
Investigation of protein-protein associations is important in
understanding structure and function relationships in living
cells. Fluorescence resonance energy transfer (FRET) based
methods are excellent tools for determining distances and
supramolecular organization of biomolecules at the cell surface
or inside the cell.
This tutorial reviews the theoretical background of FRET, its
flow cytometric and image cytometric applications giving
recipe like instructions, and provides critical evaluations of the
methods as well.
Saturday, 20 May
11:30 - 13:00
Tutorial 15: Cytometry of
• Selecting fluorophores (spectral and sterical Cell Cycle and Apoptosis ..................... Room 304
In flow cytometric FRET topics covered will include:
considerations);
• Choosing instruments (available excitation and detection);
• Autofluorescence correction for improving accuracy;
• Calibration of FRET efficiency using GFP analogs;
•· FRET determination based on anisotropy measurements.
In image cytometric FRET topics covered will include:
• Intensity based FRET imaging;
•· Donor photobleaching FRET;
• Acceptor photobleaching FRET;
• Dual FRET approach, combining donor and acceptor
photobleaching.
In the prospective part of the tutorial combination of new
probes along with new FRET modalities will be discussed.
Tutorial 14: Antigen-Specific
Cytometry ............................................ Room 303
Andreas Thiel and Mario Assenmacher
A small number of B and T cells expressing specific receptors
for a certain antigenic determinant direct the adaptive immune
response to this antigen and thus are responsible for immunity
against pathogens or tumors as well as for autoimmunity,
allergies, or transplant rejection.
However, the cytometric identification of these cells has been
difficult for many years. We will give an overview on recent
technological developments that now offer the possibility to
identify, purify, and functionally analyze antigen-specific
lymphocytes, e.g. via peptide/MHC-multimers, intracellular
cytokine staining, cytokine secretion assay, proliferation assays
and staining of CD154 or CD107a.
Zbigniew Darzynkiewicz and Frank Traganos
Cell cycle-related topics will include:
• Introduction to and description of cell cycle compartments;
• Univariate cellular DNA content cytometry
• Methods of measurement and analysis, limitations and
problems;
• Bi- and multivariate analysis using variety of markers to
identify G0, G1, S, G2 vs M cells;
• Cytometric approaches to analyze cell cycle kinetics;
• Cell synchronization, BrdU incorporation, stathmokinesis;
• Cytometry of cyclin proteins and cell cycle-associated
post-translational modifications.
Apoptosis–related topics will include:
• Characteristic features used to identify apoptotic and
necrotic cells by cytometry;
• Methods to estimate incidence of apoptosis in vitro and in
vivo;
• Methods to assess cell cycle-phase specificity of apoptosis;
• Analysis of mitochondrial changes during apoptosis;
• Assessment of DNA damage by genotoxic factors in relation
to induction of apoptosis;
• Difficulties and common pitfalls in assessment of
incidence of apoptosis. Participants will learn the most
useful methods to analyze cell cycle and measure
incidence of apoptosis. Particular attention will be given
to reveal applicability of flow- and laser scanningcytometry in mechanistic studies on protein interactions
in regulation of cell cycle progression and induction of
apoptosis.
25
Frontiers Lecture Series
Saturday, 20 May
14:45 – 16:45
Frontiers #1
2. Single Cell Kinase Signaling for Mechanistic
and Clinical Analyses .................. Room 200C
Chair: John Nolan
Garry Nolan
Intracellular assays of signaling
systems has been limited by
an inability to correlate
functional subsets of cells in
complex populations based on
active kinase states or other
nodal signaling junctions. Such
correlations could be
important to distinguish
changes in signaling status that arise in rare cell subsets during
functional activation or in disease manifestation. Simultaneous
3. Location Protemics: Image Informatics
for Systems Biology ................... Room 200C
Chair: John Nolan
Robert Murphy
Systems Biology requires
comprehensive, systematic
data on all aspects and levels
of biological organization and
function. In addition to
information on the sequence,
structure, activities, and
binding interactions of all
biological macromolecules,
the creation of accurate, predictive models of cell behavior
will require detailed information on the distributions of those
molecules within cells and the ways in which those distributions
change over the cell cycle and in response to mutations or
external stimuli. Current information on sub cellular location in
protein databases is limited to unstructured text descriptions
or sets of terms assigned by human curators. These entries do
not permit basic operations that are common to other
biological databases, such as measurement of the degree of
similarity between the distributions of two proteins, and they
are not able to fully capture the complexity of protein patterns
26
detection of activated kinases and phosphoproteins in
simultaneous pathways in subpopulations of complex cell
populations by multi-parameter flow cytometric analysis allows
identification of signaling cascades for disease states by
ordering of kinase activation and phosphoprotein status in
signaling hierarchies. Importantly, we demonstrate that ordering
of these activations require multiple interrogations of cells,
and that the networks discovered are reflective of deeper
correlations. Using Bayesian Network analysis (a form of
machine learning) one can infer pathway connectivity in an
automated fashion, allowing for high throughput derivations
of signaling system networks graphs in PRIMARY CELLS. Notably,
when kinase inhibitors, previously selected in in vitro assays
are tested on complex cell populations, single cell analysis of
signaling states reveals shocking differences in the inhibition
of kinase activity in different cell subsets that will be discussed.
The approach has powerful applications in mechanistic
understanding, drug screening, and patient stratification for
prediction of disease outcome in cancer, autoimmunity,
infection, based on signaling network status.
that can be observed. The field of location proteomics seeks to
provide automated, objective, high-resolution descriptions of
protein location patterns within cells.The initial foundation for
the field was the demonstration that automated classifiers
could be trained to recognize all major subcellular patterns in
fluorescence microscope images, and especially the critical
finding was that such systems could discriminate location
patterns better than visual examination.The very high accuracy
(over 98% on single 3D images) of these systems gave
confidence that the numerical features used to describe
location patterns could form a basis for extending the methods
to unsupervised learning of patterns. To this end, we have
described grouping of proteins into statisticallyindistinguishable location patterns using consensus clustering
methods. The resulting clusters, or location families, are
analogous to clusters found for other domains, such as protein
sequence families. Our current work is focusing on extending
this work in a number of new directions.These include analyzing
the temporal dependence of subcellular patterns, generalizing
pattern analysis across many cell types, analyzing mixed
multicell images in cultures and tissues, and creating generative
models of subcellular patterns that can be incorporated into
comprehensive models of cell behavior. The combination of
these methods with large scale, high throughput imaging
approaches will allow realistic cell modeling that reflects
detailed knowledge of the subcellular location of all proteins.
We anticipate that work in this field will also lead to improved
diagnostics based on subcellular pattern discrimination.
Sunday, 21 May
10:00 – 12:00
Frontiers #2
4. The NIH Chemical Genomics Center:
Annotating the Biological Activity of
Chemical Libraries Using Quantitative
High Throughput Screening ...... Room 200C
Jim Inglese
The NIH Chemical Genomics
Center (NCGC; www.ncgc.
nih.gov/) is the founding
member of the Molecular
Libraries Screening Center
Network (MLSCN), a network
of ten centers established as
part of the NIH Roadmap for
Medical Research (nihroadmap.
nih.gov/). The mission of the NCGC is to make accessible the
technologies and protocols of high throughput biomolecular
5. High Throughput Flow Cytometry
and the NIH Roadmap Molecular
Libraries Initiative ...................... Room 200C
Larry Sklar
The high throughput (HT) flow
cytometry platform HyperCyt
is adept at both cell and
particle-based assays and is
compatible with both high
content and multiplex analysis.
Our cell-based assays have
initially been directed against
G protein-coupled receptor
(GPCR) targets where we have identified novel small molecule
ligands for peptide and steroid receptors. We have developed
general particle-based multiplexed approaches compatible
with assemblies of soluble membrane receptors, receptor tails,
proteases, kinases, nucleases, etc. We have probed the
screening and chemistry optimization, developed primarily in
the pharmaceutical and biotech industries, to academic
investigators. The ‘product’ emerging from the NCGC pipeline
will not be drugs, but rather chemical probes to aid in the
understanding of biology and validation of therapeutic targets.
In refining the screening process, the NCGC has developed a
strategy called “quantitative HTS” (qHTS) to generate
concentration-response curves for a range of biochemical and
cellular assays on large compound collections using existing
technologies. Our process is highly refractory to false positives,
easily identifies compounds of low potency and efficacy, and
demonstrates the potential to build high-quality chemical
genomic databases. Examples from this new paradigm will be
presented.
mechanism of partial agonism and the steps in signal
transduction using flow cytometry-based kinetic analysis with
soluble GPCR. We have also developed approaches probing
cell-cell adhesion, nanoscale integrin conformational changes,
and responses to nanoscale intercellular forces.
The NIH Roadmap Molecular Libraries Initiative (MLI) has given
us the opportunity, through the New Mexico Molecular Libraries
Screening Center (NMMLSCN, http://nmmlsc/) to implement
HT flow cytometry for the international research community.
The MLSCN has expertise in: 1) target and assay development;
2) the integration of flow cytometric and virtual screening to
enhance the discovery process; and 3) medicinal chemistry for
the optimization of active molecules and the development of
imaging agents. Through MLI and collaborations with
investigators outside the MLSCN, we are currently developing
cell-based assays for cytotoxicity where both cell viability and
cell number can be recorded, bacterial virulence, multi-drug
resistance, androgen response, protein expression, and generic
cell responses, to name a few. We are also working on particlebased multiplexed protein-protein assays for interactions
between Bcl-2 family members and generic proteinoligonucleotide interactions.
27
Monday, 22 May
10:00 – 12:00
Frontiers #3
Protein Engineering by Yeast
Surface Display ........................... Room 200C
glycoproteins; and 2) quantitative measurement of phenotypes
and correspondingly precise library screening design.
Chair: Paul Smith
Yeast display has been used to: isolate novel human intibodies
and mature them to unprecendented femtomolar affinities;
develop a potent huntingtin aggregation – inhibitory intrabody;
engineer T cell receptor expressions, binding affinity and
specificity; map conformational epitopes on viral and tumor
antigens; design more potent cytokines; alter enzymatic
stereospecificity; and affinity mature integrins against their
ligands.
Dane Wittrup
Combinatorial polypeptide
libraries can be displayed on
the surface of yeast and
screened by two-color flow
cytometry to identify mutants
with improved binding affinity,
stability, or expression. The
particular advantages of yeast
display relative to phage
display are: 1) the use of a
eukaryotic host that better expresses disulfide-rich
6. The Pharmacodynamics of Molecular
Cancer Therapeutics................... Room 200C
Chair: Paul Smith
David Hedley
Our approach to cancer is
being revolutionized through
rapid progress understanding
the molecular mechanisms,
coupled to rational drug
design programs producing
highly selective agents to
target these mechanisms.
Despite
the
current
excitement, there are
formidable obstacles to making effective molecular cancer
therapeutics a clinical reality. Unlike classical chemotherapy
and radiotherapy, the drugs are highly selective in their actions,
and effective only when their molecular target is playing a
significant role driving the malignant process. Due to the
complex, multigenetic basis of cancer development, it is unlikely
that a single molecular targeted agent could achieve long term
cancer control in the clinic. More likely, optimal treatment will
consist of combinations of agents, rationally selected based on
understanding the downstream interactions of drug targets,
and individualized by analysis of the patient’s tumour tissue.
Fluorescence-based techniques using flow or image cytometry
28
have major advantages studying complex biology, because they
address the problems of cellular heterogeneity, and are able to
study molecular interactions through the use of multiple
fluorescence labels. The development of phosphospecific
antibodies has allowed the introduction of techniques to study
signal transduction at the single cell level, including the analysis
of complex signaling interactions.This is of particular importance
to molecular oncology, since a large number of agents currently
in clinical trial inhibit signaling pathways. As well as measuring
baseline activity to identify if the drug target is being expressed
in the cancer cells, cytometric methods can also be used to
monitor pharmacodynamic effects during treatment.
Pharmacodynamics is a branch of pharmacology that asks how
drugs impact on the host tissues, whereas pharmacokinetics
studies how drugs are metabolized and eliminated by the
patient. During the early phases of drug development, it is
important to show that the drug is interacting with its target in
vivo, and to determine the relationships between drug dose
and the extent of target inhibition. Along with others, our group
has been able to develop pharmacodynamic markers based
on flow cytometry and fluorescence image analysis, validate
these using experimental systems, and then translate the
methods to the actual clinical situation. Pharmacodynamic
information is critical to understanding what is going on in a
patient’s cancer cells during treatment, in order to explain why
treatment works in some patients but not in others. Although
still in its infancy, cytometry-based pharmacodynamics has the
potential to play a major role bridging between basic science,
pathology, pharmacology, and molecular oncology.
Tuesday, 23 May
10:00 – 12:00
Frontiers #4
7. Searching for an HIV Vaccine – The Role of
Flow Cytometry .......................... Room 200C
Chair: Lori Krueger
Mario Roederer
Vaccine development to
protect
against
HIV
development is actively
proceeding on two fronts:
generation of a sterilizing
(neutralizing)
humoral
response, and generation of an
effective cellular response. To
date, it has been impossible to
generate an antibody response of sufficient potency that
sterilizing vaccination is possible. Nonetheless, a vaccine can
be considered successful on a global scale if the induced cellular
response is sufficient to dampen viral loads (reducing
transmission) as well as reducing morbidity and mortality after
infection. We use a variety of adjuvants, delivery mechanisms,
and immunogens to induce a variety of T cell responses. By
challenging vaccinated nonhuman primates with live virus, we
hope to identify the kinds of responses that are best correlated
with protection as measured by a reduction in peak viral load,
set point viral load, and amelioration of the dramatic destruction
8. Human Stem Cell Biology Limitations and
Potential Applications ............... Room 200C
Chair: Lori Krueger
Mickie Bhatia
Stem cells are distinguished
from all other cell types by
their unique self renewal and
developmental properties.
Human stem cells can be
divided into two major
categories; those that come
from embryonic origins
(intercell mass of human
embryos) or those derived
of memory CD4 T cells during acute infection. As we move
forward through phase I and II clinical trials in humans, and
prepare for phase III, we are keen to determine whether or not
these types of responses are induced in humans as well.
The primary tool for determining the quantity and quality of
the T cell response is flow cytometry. Using sophisticated
instrumentation, software, reagents, and techniques, we are
able to measure five or more different functional responses
simultaneously from each cell (e.g., cytokine profile, cytotoxic
potential, proliferative capacity). These complex combinations
of functions reveal that there are a number of distinct “flavors”
of T cell responses present in either naturally infected or
vaccinated subjects; the task now is to identify which of these
flavors is most suited to protection from challenge. In addition,
by studying rapid HIV progressors vs. long term nonprogressors,
we are beginning to identify differences in T cell responses
that are correlated with disease pathogenesis, perhaps pointing
us towards desirable types of vaccine responses.
Significant challenges remain, however.The automation of the
sample analyses, the automation of the data analyses,
compliance with GLP, and validation of these procedures is an
enormous challenge. Integration and presentation of the highly
complex datasets is still intensely laborious and challenging. In
these terms, flow cytometry technology development is shifting
from the instrumentation to the software and data interpretation
aspects, which have yet to catch up to the rapid growth of the
hardware capabilities.
from adult sources and termed somatic stem cells. Both these
stem cell types are thought to have immense clinical potential
in cell replacement therapy yet each has distinct limitations.
The generation of transplantable cells of a specific lineage from
embryonic stem cells requires a detailed knowledge of
molecular and cellular development that guides differentiation
towards mature cell types. Unfortunately, our lack of
understanding of human development prevents effective
differentiation protocols to generate adequate numbers of cells
for transplantation. Adult/somatic stem cells represent lineage
specified cells with regenerative properties of specific tissues,
but lack the ex vivo expansion capacity inherent to embryonic
stem cells. Here we will discuss these comparative properties
and reveal new approaches in using adult stem cells for
regeneration of a wide variety of tissues, and the use of
embryonic stem cells as a unique model to understand early
initiation events related to human cancer.
29
Wednesday, 24 May
10:00 – 12:00
Frontiers #5 – Fulwyler Session
9. The LCOS-Based Programmable Array
Microscope (PAM) ...................... Room 200C
Chair: Marvin van Dilla
Tom Jovin
In 1998, we reported the
realization of an optically
sectioning fluorescence
microscope (PAM) for rapid,
light efficient 3D imaging of
living specimens.1,2 The design
was based on conjugate
structured illumination &
detection implemented with
a spatial light modulator (SLM)
located at an image plane of a conventional fluorescence
microscope. The major advantages of the PAM are: (1) compact
design with no moving parts; (2) very significant speedup/
improvement in sectioning due to a pixel duty cycle of up to
50%, and simultaneous detection & processing of both
conjugate (in-focus) & non-conjugate (out-of-focus) images;
(3) ultrasensitive detection via electron multiplying CCD
cameras; (4) programmable & adaptive optical sectioning based
on libraries of dot, line & pseudo-random (Sylvester) sequence
patterns; (5) flexible light sources, including LEDs; (6) generation
& detection of arbitrarily polarization patterns; (7) compatibility
with hyperspectral & lifetime-resolved imaging; (8)
incorporation of light sources for photo-destruction and/or
10. Chemical Cytometry: Analytical
Chemistry of Single Cells ........... Room 200C
Chair: Marvin van Dilla
Norm Dovichi
It is now possible to generate
one- and two-dimensional
protein electrophoresis data
from single mammalian cells.
These separations are capable
of resolving a hundred
components from the cell, and
the data can be correlated
with cell cycle and other
30
transformation, modes compatible with FRAP, FLIP & FCS/ICS;
(9) minimal photobleaching. We report here the design,
operation & application(s) of a new generation commercial
PAM created by the combined efforts of the MPIbpc (Mol Biol
Dept) and Cairn Research Ltd. (UK). The stand-alone module,
including light source(s) and detector(s), features an innovative
catadioptric design and a ferroelectric liquid-crystal-on-silicon
(LCoS) SLM instead of the original DMD. It can be attached to a
side port of a (ny) unmodified fluorescence microscope. The
Cairn prototype system currently operated at the MPI
incorporates a 6-position high-intensity LED illuminator and an
Andor iXon emCCD camera, and is mounted on an Olympus
IX71 inverted microscope with 60-150x objectives and a PZ
translator, a Cairn Optoscan monochromator, and a Prior
Scientific ProScan stage. Further enhancements are being
incorporated: (i) point- and line-wise spectral resolution,
detecting with an Applied Scientific Imaging SpectraCube
imager or a Specim ImSpector imaging spectrograph; and (ii)
lifetime imaging (FLIM) using phase-modulation2 and TCSPC
modules. Multiphoton operation and other nonlinear techniques
should be feasible.
Real-time sectioning with 50-100 ms exposures and lag-free
manual focusing has been achieved with a single LED source.
Operation is insensitive to mechanical shock applied to the
microscope table; thus, the system is suitable for ruggedized
(field) use. It is currently being applied to developmental biology
and signal transduction studies, e.g. based on quantum dot
ligands3. 1Verveer PJ et al. 1998. J. Microsc.189:192; 2Hanley QS
et al. 2005. Cytometry 67A:112 (latest PAM paper); 3Lidke DS et
al. 2005. J. Cell Biol. 170:619.
properties of the cells. Examples have been generated from
breast, prostate, colon, and esophageal cancer cell lines, from
macrophages, astrocytes and neurons, and from
osteoprecursors and myoblasts. Instrumentation is under
development that can characterize tens of thousands of cells
per day in one-dimensional electrophoresis and thousands of
cells per day in two-dimensional electrophoresis. Other
instrumentation is able to detect specific proteins at the single
copy level in single cells and determine post-translational
modifications of that protein. Finally, metabolic pathways for
both carbohydrates and glycolipids have been monitored in
single cells.
PLENARY SESSIONS
Sunday, 21 May
14:00 – 15:30
Session 1
11. Multimode Live Cell Imaging Reveals a
Novel Method of Cellular Communication
n the Immune System ................. Room 200C
Simon Watkins
Engagement of cell surface
receptors leads to intracellular
signaling that has generally
been shown to alter
phenotype and function of
individual cells. Using a variety
of cutting edge optical
imaging methods we show
here that myeloid-lineage
12. Fluorescent Speckle
Microscopy .................................. Room 200C
Gaudenz Danuser
Fluorescent
speckle
microscopy (FSM) is a method
to analyze the movement and
assembly/disassembly
dynamics of macromolecular
structures. It capitalizes on
fluorescent
analog
cytochemistry, in which
purified protein is covalently
linked to a fluorophore and microinjected or expressed as a
GFP fusion in cells. Fluorescent protein coassembles with
unlabeled, endogeneous protein, visualizing the localization
and architectural organization of the structure inside a living
cell. Despite its broad application, this classic approach has been
limited in reporting subcellular dynamics because of the
inherently high background fluorescence from unincorporated
and out-of-focus incorporated fluorescent subunits and the
uniform labeling of the target structure. However, if the labeled
subunits make up less than 1% of total pool of subunits,
fluorophore patterns of high non-uniformity accumulate.They
provide a unique random code identifying specific subareas of
dendritic cells and monocytes can be triggered to flux calcium
by mechanical contact with a microprobe, and that the signal
can be propagated within seconds to other cells at distances
up to a hundred microns away by membranous connections
called tunneling nanotubules (TNTs). These form a complex
and transient network in live cells, with individual TNTs
exhibiting great variation in length and diameter. In addition to
calcium fluxes, microinjected dye tracers can be transferred
through these connections. Following TNT-mediated
stimulation, spreading of lamellipodia occurs in dendritic cells
characteristic of that seen during the phagocytic response to
bacteria. These results demonstrate that functional signals
generated in a single cell can be transmitted to other cells
through a physically connected network.
the target structure in space and time. Addition of new
fluorophores to the code indicates the local association of
subunits; subtraction of fluorophores the local dissociation.
These molecular events can be monitored by diffraction-limited
imaging using wide-field, total internal reflection, or spinning
disc confocal fluorescence microscopy equipped with sensitive
CCD cameras. The resulting images display punctate patterns
of local maxima, called speckles, over a dimmer background.
Speckles correspond to regions of the size of the point-spreadfunction with a higher fluorophore density and serve as fiduciary
marks of the dynamics of the underlying structure. Although
conceptually simple, the interpretation of FSM time-lapse
sequences requires sophisticated computational tools for the
tracking and statistical analysis of the intrinsically stochastic
and highly convoluted signals. In this lecture I will cover the
basic design of algorithms that simultaneously track thousands
of speckles; and statistical models that allow the conversion of
positional fluctuations and speckle intensity variations into
spatiotemporally resolved maps of transport, turnover, and
viscoelasticity of the probed structure. I will then report how
quantitative FSM yielded major discoveries of the dynamic
organization of the actin cytoskeleton in migrating cells and of
the transient interaction of the cytoskeleton with other cellular
assemblies such as focal adhesions. With a second set of
examples I will indicate how we exploit the submicron
resolution of this technique to infer molecular mechanisms of
force generation during cell migration. Finally, I will illustrate
the power of FSM as a general quantitative method for imaging
the dynamics of any multiprotein structure in cells and in vitro.
31
PLENARY SESSIONS
Session 1
13. Facile Generation of Biosensors to
Study Endogenous Protein Activation
in Living Cells ............................... Room 200C
Klaus Hahn
We are developing new
methods for studying the
spatio-temporal dynamics of
signaling with minimal
perturbation. By conjugating
novel dyes to affinity reagents,
activities of endogenous
proteins can be studied with
high sensitivity.This approach
opens the door to high
throughput generation of biosensors via phage display and
other screening for biosensor affinity elements. The approach
also readily permits analysis of multiple signaling activities in
the same cell.
32
Sunday, 21 May
14:00 – 15:30
PLENARY SESSIONS
Monday, 22 May
14:00 – 15:30
Session 2
14. Multiplexed Hybrid Cytometry-Based
Application Platform Technology:
A Tool to Fight the HIV Pandemic
in the 21st Century ....................... Room 200C
Frank Mandy
In sub-Saharan Africa about 12
million people perish every
year from: HIV, malaria and
tuberculosis. For the majority
of the individuals who make
up these gruesome annual
statistics, the cause of death
is uninvestigated. The
introduction of affordable
antiretroviral therapy (ART) in parts of Africa provides an
opportunity to establish infrastructure to support laboratory
medicine. This movement is a significant step in the direction
of an effective health care system. Conventional laboratory
medicine from resource rich regions is incompatible with
African economical, cultural and political realities. In subSaharan countries 38% of the population exists on <$1 a day,
with a gross national income per capita <$500. In the past, in
most resource limited regions, founding agencies’ concentrated
15. Regulation and Therapeutic
Targeting of Human Leukemia
Stem Cells .................................... Room 200C
Kristen Hope
In acute myeloid leukemia
(AML) the leukemic clone is
organized as a hierarchy
originating from rare leukemic
stem cells (LSC). Our interest
has therefore been in
identifying and therapeutically
exploiting the molecular
mechanisms that are uniquely
required by these LSC.
on disease prevention and provision of care. Until now, little
effort has been exerted to build sustainable laboratory capacity.
With the introduction of ART in Africa, health care policy makers
and clinical scientist recognize the urgent need for affordable
and sustainable laboratory infrastructure to support the
diagnosis and treatment of HIV. For 25 years, flow cytometry
has been the CD4 T-cell enumerating devic, to monitoring
immune status of HIV infected individuals. Recently, numerous
companies introduced affordable and robust cytometers both
the flow and none-flow variety. Currently, the lower cost,
dedicated instruments are the most popular choice for CD4 Tcell counting in Africa. Accumulative sales of these instruments
are reaching significant numbers. When establishing affordable
and sustainable comprehensive laboratory infrastructure is the
overall long term objective; they in fact may turn out to be not
the most cost effective option. In this presentation features of
the hybrid flow based application platform (HyFAP) will be
covered. The focus is on how to harness HyFAP and make
economical sense in sub-Sahara Africa. They run both cell and
microsphere based assays. Evidence will be presented to
illustrate how a multi-functional and multiplexing capacity is a
realistic option in resource poor countries. In the future, HyFAP
will be connected to GSM wireless external quality monitoring
services (WEMS). The integration with WEMS will assure
minimum global standards for immunology laboratories. With
HyFAP, it is possible to build laboratory capacity to provide rapid,
accurate affordable and reliable diagnostic tests to battle
infectious diseases in most resource poor regions of the globe.
Although gene expression analysis is a common way of
identifying the molecular players in stem cell function, few
have explored the potential for microRNAs (miRNAs) in such a
regulatory role. miRNAs are 22 nucleotide (nt) non-coding RNAs
processed from hairpin precursors that regulate translational
repression of target genes. miRNAs have been implicated in
directing diverse biological processes including neoplasia.The
identification of a set of embryonic stem cell specific miRNAs
suggests that these may be involved in maintenance of the
pluripotent state while a study linking miRNAs to fate
determination showed ectopic expression of a specific miRNAs
in hematopoietic progenitors could promote B or T cell
differentiation. To address the role of miRNAs in the regulation
of LSC we performed miRNA array analysis on 4 purified
fractions based on CD34 and CD38 expression from 6 primary
AMLs. We identified a unique miRNA signature that
discriminates the CD34+ CD38- fraction from more mature
populations. One candidate, mir155, was also found to be
33
PLENARY SESSIONS
Monday, 22 May
14:00 – 15:30
Session 2
differentially highly expressed in the stem cell fraction of normal
cord blood by affymetrix array. RNAi-mediated knockdown of
mir155 in a novel CD34+ leukemic cell line resulted in a loss of
CD34 expression, an increase of differentiation antigen
expression and significantly reduced proliferative potential. Our
results are suggestive of a role for microRNAs in the regulation
of LSC and leukemogenesis. In order to cure AML, LSC must be
effectively targeted, however as existing therapeutic strategies
target cycling cells and LSC are largely quiescent, new
approaches must be found. We have shown previously that an
activating monoclonal antibody (H90) directed against the
adhesion molecule CD44 can release the AML differentiation
block when administered in vitro. To address whether CD44
activation can act at the level of the LSC, H90 was administered
to NOD/SCID mice transplanted with primary AML cells. We
show that H90 greatly impairs leukemic repopulation by up to
95% compared to mice injected with control antibody. In
addition, post-treatment grafts exhibited a much reduced level
of primitive cells and an increase in immunophenotypically
differentiated cells. The absence of leukemia in serially
transplanted mice established direct targeting of LSC. The
mechanism of H90 induced LSC loss involves both a promotion
of LSC commitment and an impairment of their homing to
supportive microenvironments.
16. Diagnosing PNH and Previously
Undiagnosed Acute Leukemias
with FLAER and Multiparameter
Flow Cytometry ........................... Room 200C
and causes lysis of normal cells but not GPI-deficient PNH cells.
FLAER is a fluorescently-labeled inactive variant of aerolysin
that does not cause lysis of cells to which it binds and this
reagent is becoming more widely used in the diagnosis of
PNH by flow. In a single tube assay, we have combined FLAER
with CD45, CD33 and CD14 that allows the simultaneous
analysis of FLAER and the GPI-linked CD14 structure on
neutrophil and monocyte lineages. Comparison to standard
CD55 and CD59 analysis shows excellent agreement.The assay
can be performed up to 48 hours after sample draw and data
interpretation is straightforward. Additionally, we were able to
detect fewer than 5% PNH-like monocytes and neutrophils in
several cases of aplastic anemia, which we were otherwise
unable to detect using CD55 and CD59 on red blood cells. Due
to the higher signal to noise ratio, the method shows increased
sensitivity in our hands over single (CD55 or CD59) parameter
analysis. Interestingly, we have also detected leukemic blasts
which show aberrant FLAER staining in several samples sent to
us for ‘PNH testing’ including 1 case each of AML-M1,
erythroleukemia (M6), a case of acute leukemia developed in
a patient with a history of CMML and two cases of acute
myelomonocytic leukemia. In all of these cases, the neutrophils
stained normally with FLAER, thereby ruling out PNH, while the
gated CD33 bright cells failed to express CD14 and bound
lower levels of FLAER.
Robert Sutherland
Paroxysmal
Nocturnal
Hemoglobinurea (PNH) is an
acquired Hematopoietic Stem
Cell disorder caused by a
somatic mutation in the Xlinked pig-a gene. This results
in a partial or absolute
deficiency of all glycophosphatidyl-inositol (GPI)linked proteins/glycoproteins. The classical approach to
diagnosis of PNH by cytometry involves the loss of at least 2
GPI-linked antigens (typically CD55 and CD59) on two different
cell lineages (RBCs and Neutrophils).The bacterial lysin Aerolysin
binds to the GPI moiety of cell surface GPI-linked molecules
34
PLENARY SESSIONS
Tuesday, 23 May
14:00 – 15:30
Session 3
17. High Content Analysis in
Drug Discovery ............................ Room 200C
Joe Trask
This talk will describe past,
current and future trends of
the use of high-content
analysis (HCA) and highcontent screening (HCS) in the
biopharmaceutical industry
and the crossover of the
technology into the academic
arena. The term HCA/HCS
18. Rare Event Detection in
Solid Cancers ............................... Room 200C
Alison Allan
Given the multi-step nature of
cancer development, there
should be several opportunities
for therapeutic targeting of
tumor cells and/or the tumor
microenvironment. The ideal
way to identify and monitor
disease progression is through
surrogate marker approaches
that are minimally invasive and allow for longitudinal testing,
such as blood tests. Our current research focuses on the
typically describes automated fluorescent imaging, image
analysis, data management and the applications thereof. The
technology not only complements flow cytometry technology,
it resembles flow cytometry during its infancy with many
challenges and a rapidly changing future. The impact ofHCA/
HCS technology in the biopharmaceutical industry is being felt
throughout the entire drug development process from early
drug discovery, target identification, target validation, screening
through preclinical biomarker discovery including toxicology
and mechanism of action studies. The technology has crosspollinated into many areas of science including basic science
and even material sciences. The goal of the talk is to provide
the audience with a brief history of HCA/HCS and case studies
where the technology has made an impact.
development of such approaches, in particular rare event
detection by image and flow cytometric methods. Identifying
rare populations requires a different approach than standard
cytometry techniques, which rely mainly on positive and
negative decisions made in either one, or at most, two
dimensional space. Recent interest in identification and
quantification of rare cell populations such as circulating
epithelial tumor cells and circulating endothelial cells in cancer
patients has pushed the limits of detection even further. Flow
and image cytometric methods are now being developed to
identify cellular populations with frequencies as low as 1-20
cells/ml. This presentation will discuss the issues that must be
addressed when designing an assay to accurately detect rare
populations of cells in blood or bone marrow, with emphasis
on detection of circulating endothelial cells and circulating
tumor cells in patients with solid tumors and in experimental
mouse models of cancer.
35
PLENARY SESSIONS
Tuesday, 23 May
14:00 – 15:30
Session 3
19. Image-Based Screen for Cell Cycle
and Cancer Targets ...................... Room 200C
Dan Rines
Chromosome segregation
during mitosis depends on the
proper function of specialized
structural and cytoskeletal
machinery. The duplicated
chromosomes are separated
equally to daughter cells by the
highly dynamic fibers of the
mitotic spindle called
microtubules. The spindle consists of a bipolar array of
microtubules where the extreme ends of the spindle are each
anchored to a centrosome. Kinetochores are large protein
complexes that assemble onto centromeric DNA sequences
and physically attach the replicated chromosomes to the
spindle fibers. Ultimately, the maintenance of genomic integrity
depends on the proper attachment of chromosomes to the
spindle and on the generation of opposing tensile forces that
pull the chromosomes apart. Failure in either of these processes
leads to unequal partitioning of the genome. However, little is
36
known about how chromatid-microtubule attachment is
mediated, or how opposing forces are generated.
Currently, two anti-cancer agents, paclitaxel (taxol) and
camptothecin, are used in the treatment of various forms of
the disease. Taxol promotes irreversible polymerization of
microtubules, disrupting their inherent dynamic nature.
Camptothecin functions by inhibiting topoisomerase I activity
and leads to large scale chromosome breakage when opposing
tensile forces are applied. The diverse action of these two antimitotic compounds and the mechanical complexity of the
segregation process suggest that many protein components
are involved. In fact, recent studies in more genetically tractable
organisms such as the budding yeast, S. cerevisiae, have
identified over 50 proteins involved in the chromatidmicrotubule attachment process alone and many of the
functional orthologues have yet to be identified in humans.
Using a RNA library of 49,000 double-stranded (ds)RNA
targeting approximately 24,000 genes, we performed a lossof-function screen for essential mitotic chromosome
segregation genes. We identified novel genes whose
inactivation caused mitotic arrest. Multi-parametric analysis of
image-based data derived from a high-content screen including
phospho-histone H3 levels, cellular proliferation and nuclear
morphology allowed us to isolate both checkpoint and
independent segregation genes.
PLENARY SESSIONS
Wednesday, 24 May
14:00 – 15:30
Session 4
20. A Human Protein Atlas for Normal
and Cancer Tissues ...................... Room 200C
Mathias Uhlen
Antibody-based proteomics
provides a powerful approach
for the functional study of the
human proteome involving
the systematic generation of
protein-specific affinity
reagents. We have used this
strategy to construct a
comprehensive, antibody-
21. Flow and Image Cytometric
FRET for Detecting Protein
Associations ................................. Room 200C
János Szöllosi
áá
Supramolecular organization
of biomolecules at the cell
surface or inside the cell has
an important role in
determining the function and
integrity of cells. Specific
techniques are available now
for detecting molecular
proximity and interactions in
cells, such as flow or image
cytometric variations of
fluorescence resonance energy transfer (FRET).
Flow cytometric techniques offer the advantage of rapid
analysis on a large number of cells (~105 cells in some minutes)
with a high statistical accuracy and a possibility for analyzing
heterogeneity at the population level. Flow cytometry,
however, does not provide any information about the spatial
localization of fluorescent probes, but instead measures the
fluorescence intensity averaged over each cell. In contrast,
microscopic techniques provide a high spatial resolution:
conventional fluorescence microscopies have a ~250 nm
resolution limited by diffraction of the optics. Although
microscopies have several further advantages in detecting
based protein atlas for expression and localization profiles in
48 normal human tissues and 20 different cancers (1). The
Human Protein Atlas is publicly available (www.proteinatlas.org)
and contains, in the first version, approximately 400,000 highresolution images corresponding to more than 700 antibodies
towards human proteins. Each image has been annotated by
certified pathologists to provide a knowledge base for
functional studies and to allow queries about protein profiles
in normal and disease tissues (2). Our results suggest it should
be possible to extend this analysis to the majority of all human
proteins thus providing a valuable tool for medical and
biological research, in particular for biomarker analysis in various
patient cohorts.
molecular dynamics of changes in the distribution or intensity
of fluorescent probes, they suffer from a low statistical reliability,
especially in the case of quantitative measurements. Thus, a
combined application of flow and image cytometry in resolving
particular biological questions can be a very powerful approach.
In flow cytometry we applied fluorescent probes with longer
wavelength excitation and multiple wavelength detection in
the emission regions so that autofluorescence correction could
be performed on a cell by cell basis in FRET analysis. These facts
improved the accuracy of the FRET method and cells with low
receptor expression, such as HPB-ALL cells transfected with
various CD45 isoforms were amenable to FRET investigation.
Combination of various forms of flow and image cytometric
FRET methods revealed distinctive expression and association
pattern of ErbB receptor tyrosine kinases on the surface of
various cancer cell lines sensitive or resistant to trastuzumab
(Herceptin®). Simultaneous application of image cytometric
FRET methods based on donor and acceptor photobleaching
provided a useful dual FRET approach revealing a unique
coassociation pattern of integrins, CD44 and ErbB2 on the surface
of tumor cells. By measuring the distances between various
monoclonal antibody epitopes on ErbB2 molecules and the
distances between epitopes and the cell membranes useful
information was provided for positioning the extracellular
domain in molecular modeling the nearly full length ErbB2
dimer. In this model favorable dimerization interactions were
predicted for the extracellular, transmembrane and protein
kinase domains, which may act in coordinated fashion in ErbB2
homodimerization, and also in heterodimers of ErbB2 with
other members of ErbB family.
37
PLENARY SESSIONS
Wednesday, 24 May
14:00 – 15:30
Session 4
22. Do Not Mind the Gap: Protein
Trafficking between the Endoplasmic
Reticulum and the Golgi Apparatus
in Plant Cells ................................ Room 200C
Federica Brandizzi
Secretory materials are
synthesized on the surface of
the endoplasmic reticulum
(ER). They are then shipped
from the ER to the Golgi
apparatus to be sorted either
back to the ER or to distal
secretory compartments such
as vacuoles and plasma
membrane. It is vital for a cell to regulate protein transport
between these organelles. The ER and Golgi are closely
associated in plant cells. How these two organelles
communicate with each other in plant cells is an important
question that remains largely unanswered.
38
To provide further understanding of the regulation of protein
export from the plant ER, we have explored the mechanisms
of protein trafficking between the ER and the Golgi apparatus
using live cell imaging techniques. It appears that plant cells
contain multiple mobile Golgi stacks distributed over the entire
cytoplasm. These stacks move with the ER by means of actinmyosin motors. The domains of the ER dedicated to the export
of proteins, the ER export sites (ERESs), form secretory units
that move along the surface of the ER together with the Golgi
stacks. We also found that the integrity of Golgi and ERESs is
regulated by the activity of specific GTPases, such as Sar1 and
Arf1.
Finally, we determined the existence of a stringent signalregulated mechanisms for ER export of multispanning, type I
and type II membrane proteins6. For example, we found that
mutations of a specific di-acidic motif (DXE) in the cytosolic tail
of proteins such as CASP, a Golgi matrix protein with a type II
membrane topology, cause a reduction of the export of this
protein from the ER. ER export of type I and multispanning
membrane proteins appears to be similarly regulated.
Our results indicate that in plant cells the ER and Golgi form a
dynamic membrane system whose components continuously
cycle through the ER via a regulated membrane trafficking
pathway.
Parallel Session 1
Advanced Microscopy and Image
Acquisition 1 ....................................... Room 301
08:00 - 09:30
Chairs: Attila Tarnok and Damir Sudar
23. COHERENT ANTI-STOKES RAMAN SCATTERING (CARS)
MICROSCOPY FOR LABEL-FREE, CHEMICALLY-SELECTIVE
BIOMEDICAL IMAGING
Conor L. Evans; Jeanette Kurian; Eric O. Potma; Mehron
Pourgish’Haag; Daniel Cote; Charles P. Lin; X. Sunney Xie
24. QUANTITATIVE LIVE IMAGING DESCRIBES
MORPHOGENETIC NUCLEAR MOVEMENTS IN EARLY
DROSOPHILA EMBRYO
Cris Luengo; Soile Keränen; Charless Fowlkes; Gunther
Weber; Min-Yu Huang; Oliver Rübel; Bernd Hamann; Damir
Sudar; Jitendra Malik; Mark D. Biggin; David W. Knowles
25. ACQUIRING MULITPLE IMAGES OF LARGE TISSUE
SECTIONS IN BOTH FLUORESCENCE AND TRANSMITTED
LIGHT USING THE LASER SCANNING CONFOCAL
TISSUESCOPE
Trudey Nicklee; David Hedley
26. COMPARISON OF FLUORESCENTLY AND CHROMATIC
LABELED TISSUE MICROARRAYS ANALYZED BY LASER
SCANNING CYTOMETRY
Ed Luther
27. HYPERCHROMATIC CYTOMETRY PRINCIPLES FOR
CYTOMICS BY SLIDE-BASED CYTOMETRY
Attila Tárnok; Mittag Anja; Wiebke Laffers; Dominik Lenz;
Andreas Gerstner
28. A MULTIMODE ENDOSCOPE FOR SIMULTANEOUS
MACROSCOPIC AND MICROSCOPIC IMAGING OF TISSUES
IN VIVO
Silas J. Leavesley; Bartlomiej Rajwa; J. Paul Robinson
Image Processing and Analysis 1 ....... Room 302
08:00 - 09:30
Chairs: Jeff Price and Tom Mistelli
29.TOPOLOGY PRESERVING STACS SEGMENTATION OF
PROTEIN SUBCELLULAR LOCATION IMAGES
Amina Chebira; Gowri Srinivasa; Lionel Coulot; Heather
Kirschner; Jose M. F. Moura; Jelena Kovacevic; Elvira Garcia
Osuna; Robert F. Murphy
30. TWO AND THREE DIMENSIONAL SEGMENTATION OF
WHOLE CELLS AND CELL NUCLEI IN TISSUE
Dean P. McCullough; Daniel Baggett; Stephen J. Lockett
31. A GENERAL TECHNIQUE FOR SEGMENTATION OF
INDIVIDUAL CELLS IN LIGHT MICROGRAPHS
Zachary Pincus; Julie A. Theriot
32. FILO: AN UNBIASED SPATIAL ANALYSIS OF FISH SIGNALS
IN INTERPHASE NUCLEI
Prabhakar Reddy Gudla; Addison Z. Yee; Takumi Takizawa;
Tom Misteli; Stephen J. Lockett
Sunday, 21 May
33. IMAGE ANALYSIS AND METADATA PROCESSING FOR CELL
STRUCTURE AND FUNCTION DESCRIPTION. EXAMPLES OF
APPLICATION AS A NEW DIAGNOSTIC APPROACH IN
THREE DIFFERENT FIELDS: HAEMATOLOGY,TOXICOLOGY
AND MICROBIOLOGY
Maria Cristina Albertini; Marco Rocchi; Augusto Accorsi;
Laura Teodori
34. NONINVASIVE FORWARD-SCATTERING SYSTEM FOR RAPID
DETECTION, CHARACTERIZATION, AND IDENTIFICATION
OF LISTERIA COLONIES. IMAGE-PROCESSING AND
ANALYSIS.
Bulent Bayraktar; Padmapriya P Banada; J. Paul Robinson;
Arun K. Bhunia; E. Daniel Hirleman; Bartlomiej Rajwa
Flow Instrumentation 1 ...................... Room 303
08:00 - 09:30
Chairs: Andrea Cossorizza and Ryan Brinkman
35. ENHANCED DIFFERENTIATION MODULE (EDM) FOR
CLINICAL FLOW CYTOMETRY ANALYSIS
J. Paul Robinson; Kathy Ragheb; Cheryl Holdman; Valeri
Patsekin; Christakis Christodoulou; Bill Kiroviac; Paul
Church; Todd Lary
36. SORTING OF ULTRA-SMALL VESICLES FOR PROTEOMIC
ANALYSIS
James N. Higginbotham; Zheng Cao; Thomas J. Utley;
James E. Crowe; Robert J. Coffey
37. A NOVEL SOFTWARE ARCHITECTURE THAT ALLOWS THE
USE OF WINLIST FOR REAL-TIME DATA ACQUISITION AND
SORTING CONTROL OF A HIGHLY AUTOMATED PARALLEL
SORTING (HAPS)
Gary Durack; Mark Hubbard; Jeremy Hatcher; C. Bruce
Bagwell
38. CLASSIFYING CELL SIGNALING PROFILES IN LEUKEMIA
Nikesh Kotecha; Jonathan Michael Irish; Garry P. Nolan
39. CLUSTER AND PRINCIPAL COMPONENT ANALYSIS FOR
THE IDENTIFICATION OF COMPLEX PHENOTYPES
Enrico Lugli; Marcello Pinti; Leonarda Troiano; Milena Nasi;
J. Paul Robinson; Valery Patsekine; Caterina Durante;
Gianfranco Salvioli; Marina Cocchi; Andrea Cossarizza
40. BIOINFORMATICS DATA STANDARDS FOR FLOW
CYTOMETRY
Ryan R. Brinkman; Josef Spidlen; Adam S. Treister; Clayton
Smith; Michael B. Ochs; Robert Gentleman; Charles
Schmitt; Perry Haaland
Cell Physiology 1 ................................. Room 304
08:00 - 09:30
Chairs: Paul Smith and Laura Teodori
41. PLASTICITY OF THE TUMOUR CELL GLYCOCALYX
PaulJ Smith; Emeline Furon; Sally Chappell; Marie
Wiltshire; RobertA Falconer; Laurence Patterson; Andrew
Goater; David Morris; Julian P.H. Burt; Rachel J. Errington
39
42. LIPID RAFTS REGULATE PDGFR CLUSTERING, ACTIVATION,
INACTIVATION AND DOWNSTREAM SIGNALING IN A CELL
CONFLUENCE-DEPENDENT MANNER
György Vereb; László Ujlaky-Nagy; János Szöllõsi
Calibration and Standardization ....... Room 202
43. DEVELOPMENT OF A NEW METHODOLOGY AND TOOLS
FOR PREDICTIVE ANALYSIS OF CYTOMIC DATA: FINDING
COMBINATIONS OF IMMUNOPHENOTYPIC PARAMETERS
WHICH CAN PREDICT THE OUTCOME OF SEPTIC SHOCK
PATIENTS
Jorge Monserrat; Eduardo Reyes; Alfredo Prieto; Angela
Hernández; Hugo Barcenilla; Raul De Pablo; David Diaz;
Cristina Sánchez; Guillermo Revilla; Melchor Álvarez-Mon
47. COMPREHENSIVE CHARACTERIZATION OF FLOW
CYTOMETER FLUORESCENCE MEASUREMENT WITH A
MINIMAL BEAD SET
Robert A. Hoffman; Joseph Trotter; Alan Stall
08:00 - 09:30
Chairs: Robert Zucker and Ted Young
48. INITIAL RESULTS FROM NATIONAL SURVEY OF Q AND B
VALUES
Eric Chase; Raymond Lannigan
44. STATIC MAGNETIC FIELDS STIMULATE SKELETAL MUSCLE
DIFFERENTIATION
Dario Coletti; Maria Cristina Albertini; Sergio Adamo; Laura
Teodori
49. DATA INTEGRITY AND REPEATABILITY IN CYTOMETRIC
MEASUREMENTS
William Ortyn; David Basiji; Brian Hall; Richard Bauer;
Cathleen Zimmerman; David Perry; Keith Frost; Richard
Esposito; Thaddeus George; Philip Morrissey
45. A SYSTEMATIC APPROACH TO ANALYZING CHANGES IN
PROTEIN SUBCELLULAR LOCATION DURING THE CELL
CYCLE
Elvira Garcia Osuna; Margaret H. Fuhrman; Jonathan W.
Jarvik; Robert F. Murphy
50. ABSOLUTE FLUORESCENCE CALIBRATION: THEORY AND
PRACTICE
Ian Theodore Young; Yuval Garini; Bart J. Vermolen; Guus
Liqui Lung
46. EVALUATION OF ANTI-CANCER TARGETS ON CIRCULATING
TUMOR CELLS TO PREDICT THERAPEUTIC SUCCESS
Arjan Tibbe; Joost Swennenhuis; Gerald Doyle; Dave
Chianese; Jan Keij; Chandra Rao; Mark Connelly; John
Verrant; Leon W.M.M. Terstappen
Parallel Session 2
Advanced Microscopy and Image Acquisition 2
Room 301
08:00 - 09:30
Chairs: Howard Shapiro and Belan Molnar
53. IMPROVING SINGLE MOLECULE FÖRSTER RESONANT
ENERGY TRANSFER MEASUREMENTS BY PULSED
INTERLEAVED EXCITATION AND FLUORESCENCE
CORRELATION SPECTROSCOPY
Steffen Rüttinger; Benedikt Kraemer; Martin Roos;
Eberhard Hildt; Felix Koberling; Rainer Macdonald
54. EXTENDED DEPTH OF FIELD IMAGING WITH THE
IMAGESTREAM EDF IMAGING FLOW CYTOMETER SYSTEM
William Ortyn; David Basiji; Keith Frost; Richard Bauer;
Richard Esposito; Cathleen Zimmerman; David Perry;
Philip Morrissey; Thaddeus George; Brian Hall
55. ABOUT A NOVEL LIGHT MICROSCOPE ARCHITECTURE
Rainer Uhl
56. CELL AND TISSUE RELATED SCANNING FLUORESCENT
VIRTUAL MICROSCOPY USING A DEDICATED DESK TOP
SCANNER SYSTEM
Bela Molnar; Viktor Sebestyen Varga; Attila Tagscherer;
Tibor Virag; Viktor Kamaras; Zsolt Tulassay
40
51. QUANTITATIVE CHARACTERIZATION OF CONFOCAL
MICROSCOPE PERFORMANCE
Edward H. Cho; StephenJ. Lockett
52. MEASUREMENT OF STABLITY AND PRECISION OF LIGHT
DETECTION IN OPTICAL MICROSCOPY
Tytus Bernas; Elikplimi Kwaku Asem; J. Paul Robinson;
Bartlomiej Rajwa
Monday, 22 May
57. AFFORDABLE CYTOMETRY FOR INFECTIOUS DISEASE
DIAGNOSIS AND MONITORING
Howard Shapiro; Nancy Perlmutter
58. ENABLING REPETITIVE PROLONGED MEASUREMENTS OF
NON-TETHERED NON-ADHERENT INDIVIDUAL CELLS IN A
MICROTITER PLATE
Mordechai Deutsch; Naomi Zurgil; Elena Afrimzon; Yana
Shafran; Assaf Deutsch
08:00 - 09:30
Chairs: Robert Murphy and Elliot Botnivick
59. THE SHAPE OF THINGS TO COME: QUANTITATIVE ANALYSIS
OF CELL MORPHOLOGY
Zachary Pincus; Natalie A. Dye; Kinneret Keren; Julie A.
Theriot
60. BUILDING GENERATIVE MODELS OF SUBCELLULAR
LOCATION PATTERNS
Ting Zhao; Robert F. Murphy
61. IMAGE CYTOMETRY PROFILING OF CELL CYCLE
PHENOTYPES IN GENOME-WIDE siRNA KNOCKDOWN
CELLS
Yan Feng; Jonathan Hoyt; Yong-Chuan Tao; Timothy
Mitchison
62. IMAGE ANALYSIS FOR STRUCTURAL GENOMICS REVEALS
NOVEL PROCESSES IN TUMOR PROGRESSION AND
APOPTOSIS
Bart J. Vermolen; Ian Theodore Young; Sabine Mai; Sherif
Louis; Vered Raz; Yuval Garini
74. A COMPLEX DIETARY SUPPLEMENT DRAMATICALLY
REDUCES RADIATION-INDUCED CHROMOSOME
ABERRATIONS, 8-OHDG LEVELS AND H2AX FOCI IN MICE
EXPRESSING ELEVATED FREE RADICAL PROCESSES
Jennifer A. Lemon; C. David Rollo; Douglas R. Boreham
63. MICRONUCLEI FORMATION, MORE THAN MEETS THE EYE?
A MULTIPARAMETRIC HIGH CONTENT ANALYSIS STUDY
USING LASER SCANNING CYTOMETRY
Raffi Manoukian; Satin Sawant; Gloria Juan
75. STATIC MAGNETIC FIELDS MODULATE CHEMICAL/
PHYSICAL INDUCED APOPTOSIS AND DNA DAMAGE BUT
NOT OXIDATIVE STRESS IN GLIOBLASTOMA PRIMARY
CELLS
Claudio Panzarella; Maria Giovanna Valente; Gianluca
Vigiliano; Donatella Tirindelli; Maria Cristina Albertini; Luigi
Campanella; Mario Barteri; Cecilia Costanza; Natale
Santucci; Laura Teodori
64. HISTONE HYPERACETYLATION CAUSES REPOSITIONING
OF CENTROMERES AND TOPOLOGICAL REORGANISATION
OF CHROMATIN IN HUMAN PROSTATE CANCER
Carla Toland; Vicky Kyle; Perry Maxwell; Peter Hamilton
08:00 - 09:30
Chairs: Steve Graves and Gray Durack
65. LIGHT EMITTING DIODES (LEDS) AND RED DIODE LASERS
FOR LOW COST MULTICOLOR FLOW CYTOMETRY
Robert A. Hoffman; David W. Houck
66. LOW COST LIGHT SOURCE AND MINIATURE DETECTORS
YIELD HIGH PERFORMANCE IN A SLOW-FLOW SYSTEM
Robert Habbersett; Jimmy Parson; Steven Graves
67. LOW COST HAND PORTABLE FLOW CYTOMETRY
Steven W. Graves; Gregory Kaduchak; Gregory R. Goddard;
Robert C. Habbersett; Michael D. Ward; John C. Martin;
Mark Naivar
68. FAST AND PARALLEL MICROFLUIDIC OPTICAL SORTERS
ENABLE SAFE AND QUICK PURIFICATIONS OF RARE CELLS
Ruud Hulspas; Manish Deshpande; John Gilbert
69. DESIGN OF A HIGH-THROUGHPUT BIOMEMS
MICROFLUIDIC CYTOMETER/SORTER
James F. Leary; Rashid Bashir
70. A HIGHLY AUTOMATED PARALLEL SORTING (HAPS)
SYSTEM THAT PROCESSES 2.5 X 109 CELLS PER HOUR
UNDER THE CONTROL OF A SINGLE OPERATOR
Gary Durack; Paul Weiss
08:00 - 09:30
Chairs: János Szöllösi and Bill Telford
71. FLOW CYTOMETRIC MEASUREMENTS OF OXIDATIVE
STRESS IN HEMOGLOBINOPATHIES
Fibach Eitan; Johnny Amer
72. ESSENTIAL REQUIREMENT OF REDUCED GLUTATHIONE
FOR THE ANTI-OXIDANT EFFECT OF THE FLAVONOID
QUERCETIN
Roberta Ferraresi; Erika Roat; Leonarda Troiano; Enrico
Lugli; Chiara Giovenzana; Maria Garcia Fernandez; Elisa
Nemes; Milena Nasi; Marcello Pinti; Andrea Cossarizza
76. SIMULTANEOUS ANALYSIS OF MULTIPLE CASPASE
ACTIVITIES IN MOUSE T LYMPHOCYTES BY FLOW
CYTOMETRY
William G. Telford; Beverly Z. Packard; Akira Komoriya
Rare Event Detection and Stem Cell
Technologies ....................................... Room 202
08:00 - 09:30
Chairs: Mike Keeney and Phil McCoy
77. DETECTION OF MIGRATING FIBROBLASTS IN THE
PERIPHERAL BLOOD BY SLIDE BASED CYTOMETRY
Ulrich Sack; Christian Eimermacher; Anja Mittag; Attila
TáRnok
78. PRACTICAL STRATEGIES FOR SUCCESSFUL RARE EVENT
DETECTION AND ISOLATION OF HUMAN HEMATOPOIETIC
STEM CELL POPULATIONS WITH FLOW CYTOMETRIC
INSTRUMENTATION
Lora W. Barsky; Ewa Zielinska; Mary A. Price; KimberlyJ
Payne; Yanjia Zhang; Qian-Lin Hao; Yuhua Zhu; Yasmin K.
Parrish; KennethI Weinberg; Gay M. Crooks
79. INHERENT LIMITATIONS IN RARE CELL DETECTION
Arjan Tibbe; Craig Miller; Leon Terstappen
80. UNEXPECTED HIGH COUNTS OF MATURE CIRCULATING
ENDOTHELIAL CELLS IN HEALTHY INDIVIDUALS
Michiel Strijbos; Jaco Kraan; Michael Den Bakker; Bart
Lambrecht; Stefan Sleijfer; Jan-Willem Gratama
81. SP CELLS, ANTHRACYCLINE UPTAKE, AND FLUORESCENCE
POLARIZATION
Timothy W. Petersen; Allan Kachelmeier; Sherrif Ibrahim;
Ger Van Den Engh
82. FLOW CYTOMETRIC ANALYSIS AND PURIFICATION OF
NEURAL AND NEURONAL CELL POPULATIONS DERIVED
FROM HUMAN EMBRYONIC STEM CELLS
Jan Pruszak; Kai-Christian Sonntag; Joris Van Arensbergen;
Takahito Yoshizaki; Moe Hein Aung; Rosario SanchezPernaute; Ole Isacson
73. MITOCHONDIRAL COMPLEX I INHIBITOR ROTENONE
INDUCES CELL DEATH BY ENHANCING REACTIVE OXYGEN
SPECIES GENERATION AND PEROXYNITRITE-INDUCED
CYTOTOXICITY IN HL-60 CELL
Jia Liu; J. Paul Robinson
41
Parallel Session 3
08:00 - 09:30
Chairs: Yuval Garini and Anne Carpenter
83. DETECTION AND ASSESSMENT OF CERVICAL
INTRAEPITHELIAL NEOPLASIA (CIN) LESIONS BY DNA
IMAGE CYTOMETRY
Sun Xiaorong
84. CELL SIZE, SHAPE AND MEMBRANE ARE TARGETS OF
STATIC MAGNETIC FIELDS AS DEMONSTRATED BY
ELECTRON, OPTIC AND ATOMIC FORCE MICROSCOPY IN
HUMAN GLIOBLASTOMA CELLS
Laura Teodori; Maria Cristina Albertini; Francesco
Uguccioni; Elisabetta Falcieri; Marco Rocchi; Antonio
Bergamaschi; Andrea Magrini; Raffaele Mucciato; Augusto
Accorsi
85. IMAGING THE CELLULAR RESPONSE TO FLUID SHEAR BY
APPLYING ULTRA-LOCALIZED FLOW FIELDS GENERATED
BY ROTATING LASER-TRAPPED MICROSPHERES
Elliot Botvinick; Gregor Knoener; Michae lW. Berns; Halina
Rubinsztein-Dunlop
Tuesday, 23 May
92. QUANTITATIVE ANALYSIS OF THERAPEUTIC MONOCLONAL
ANTIBODY LOCALIZATION TO ENDOSOMES AND
LYSOSOMES USING THE IMAGESTREAM IMAGING FLOW
CYTOMETER.
Brian Hall; Philip Morrissey; Che-Leung Law; Kristine
Gordon; David Lynch; Keith Frost; Thaddeus George; David
Basiji; Cathleen Zimmerman; William Ortyn; Richard
Bauer; David J. Perry; Richard Esposito
93. DEVELOPMENT OF HIGHLY-SECRETING
BIOPHARMACEUTICAL CELL LINES VIA IN SITU
MEASUREMENT OF CELL-SPECIFIC ANTIBODY SECRETION,
LASER-MEDIATED CELL PURIFICATION, AND AUTOMATED
CLONE TRACKING AND ANALYSIS
Elie Hanania; Janine Stevens; Gary Bright; Manfred Koller
94. DETERMINATION OF IN VIVO TOXICITIES OF GAMMASECRETASE INHIBITORS IN SPLEEN MARGINAL ZONE B
CELLS BY FLOW CYTOMETRY
Sharon Ann Sokolowski; Barbara-Anne Martin; AnneM.
Ryan; Gary B. Freeman; Carol D. Hicks; Lit-Fui Lau; Nikolay
Pozdnyakov
86. APPLICATION OF QUANTITATIVE MORPHOLOGICAL
CYTOMETRY FOR EVALUATION OF SHEAR STRESS
Dominik Lenz; Bulent Bayraktar; Silas Leavesley; J. Paul
Robinson; Bartlomiej Rajwa
87. PROTEIN SUBCELLULAR LOCATION IMAGE DATABASE FOR
COMPREHENSIVE IMAGE RETRIEVAL AND AUTOMATED
INTERPRETATION
Juchang Hua; Ting Zhao; Shann-Ching Chen; Yanhua Hu;
Amol Shanbhag; Justin Newberg; Swapnil Upganlawar;
RobertF. Murphy
95. NON-INVASIVE AND LABEL-FREE FLOW CYTOMETRY
Marco Di Berardino; Grit Schade; Adrian Huwiler; Thomas
Hessler
88. CELLPROFILER: FREE, HIGH-THROUGHPUT SOFTWARE FOR
AUTOMATICALLY MEASURING CELLS IN IMAGES
Anne E. Carpenter
BioPharma Applications..................... Room 302
08:00 - 09:30
Chairs: Phil Marder and Padma Narayanan
89. DEVELOPMENT OF MULTILAYERED NANOPARTICLE
SYSTEMS FOR NANOMEDICINE
James F. Leary; TarlW Prow; Donald Eugene Bergstrom
90. CYTOMETRY AS AN AID IN DEVELOPMENT OF
ANTIMICROBIAL AGENTS
Howard Shapiro; Nancy Perlmutter
91. DEVELOPMENT OF A PHOSPHATIDYLINOSITOL 3-KINASE
DRUG ACTIVITY BIOMARKER ASSAY IN PLATELETS
Rita Bowers; Philip Marder; Lisa Green; Andrew Faber;
Phillip Schwier; Candice Horn; James Thomas
42
08:00 - 09:30
Chairs: John Nolan and Dave Basiji
96. MULTISPECTRAL CYTOMETY: A POWERFUL NEW
TECHNOLOGY IN CELL ANALYSIS
J. Paul Robinson; Bartlomiej Rajwa; Kathy Rajheb; JamesT.
Jones; Gérald Grégori; Valery P. Patsekin
97. SPECTRAL ANALYSIS FLOW CYTOMETER: MODULAR
INCLUSION OF HIGH RESOLUTION SPECTRAL ANALYSIS
Gregory Goddard; John Martin; Mark Naivar; Peter
Goodwin; Steven Graves; Robert Habbersett; John P.
Nolan; James Jett
98. RAMAN FLOW CYTOMETRY
John P. Nolan; Dakota Watson; Daniel Gaskill; Mirianas
Chachisvilis; Steven Graves; Lief Brown; Stephen Doorn;
Hicham Fenniri
99. FLUORESCENCE SENSITIVITY AND COMPENSATION IN
MULTISPECTRAL FLOW IMAGING
David Basiji; William Ortyn; Cathleen Zimmerman; David
Perry; David Coder; Keith Frost; Brian Hall; Thaddeus
George; Richard Esposito; Richard Bauer; Philip Morrissey
100. MODULAR, EXPANDABLE, HIGH-SPEED DIGITAL DATA
ACQUISITION SYSTEM DESIGNED TO SUPPORT MIXED
MODE DATA COLLECTION FROM PMTS, PHOTONCOUNTING APDS, AND HIGH-RESOLUTION CCD ARRAYS
Mark Naivar; James Jett; Jimmy Parson; Robert
Habbersett; Steven Graves; John Martin; Mark Wilder; John
P. Nolan; James Freyer
Immune Monitoring ........................... Room 202
08:00 - 09:30
Chairs: David Hedley and Leon Terstrappen
08:00 - 09:30
Chairs: Mario Roederer and Jan Gratama
101. CORRELATED MULTIPARAMETER FLOW CYTOMETRY AND
MICROSCOPY DEMONSTRATE THAT THE RARE MOUSE
SMALL INTESTINAL EPITHELIAL PROGENITORS
EXPRESSING NEUROGENIN 3 ARE SLOWLY CYCLING CELLS
DERIVED FROM A BIPOTENTIAL PRECURSOR AND GIVE
RISE TO ENTEROENDOCRINE CELLS
Matthew Bjerknes; Hazel Cheng
107. QUANTITATIVE ANALYSIS OF ANTIGEN-SPECIFIC
ANTIBODY RESPONSES
Henri Van Der Heyde; WilliamP. Weidanz; James Burns;
Irene Gramaglia; John P. Nolan
102. THE RELATIONSHIP BETWEEN DIFFERENT NEOPLASTIC
CERVICAL LESIONS AND THE FREQUENCY OF ANEUPLOID
CELLS WITH DNA AMOUNT GREATER THAN 5C
Wang Jian
103. COMPARISON OF FLUORESCEIN AND PHYCOERYTHRIN
CONJUGATES FOR QUANTIFYING CD20 EXPRESSION ON
NORMAL AND LEUKEMIC B-CELLS
Gerald Marti; Lili Wang; Fatima Abbasi; Adolfas Gaigalas;
Robert F. Vogt
104.TRASTUZUMAB AND PERTUZUMAB DIFFERENTIALLY
AFFECT HER2/NEU OVEREXPRESSING BREAST CANCER
CELLS
Simone Diermeier; Arabel Vollmann; Andrea Sassen;
Ferdinand Hofstaedter; Gero Brockhoff
105. APPLICATION OF RATIOMETRIC CELL ENUMERATION TO
THE DEVELOPMENT OF FLOW CYTOMETRY CELL
CYTOTOXICITY ASSAYS
David Diaz; Alfredo Prieto; Hugo Barcenilla; Jorge
Monserrat; Luis Chara; Julio Chevarria; Miguel Ángel
Sánchez; Eduardo Reyes; Melchor Álvarez-Mon
106.THE INVOLVEMENT OF LYSOPHOSPHATIDYLECHOLINE
(LPC) IN LYMPHOCYTE APOPTOSIS IN ATHEROSCLEROSIS
Elena Afrimzon; Naomi Zurgil; Yana Shafran; Mordechai
Deutsch
Parallel Session 4
Image Spectral Analysis ..................... Room 301
08:00 - 09:30/
Chairs: Robert Leif and Richard Levensen
113. INCREASING THE LUMINESCENCE OF LANTHANIDE(III)
COMPLEXES
Robert Cary Leif; Sean Yang; Alfred Bromm; Margie C.
Becker; Lidia M. Vallarino
114. LIPID INTERACTION AND LOCALIZATION WITH NILE RED
BY FRET MULTIPHOTON CONFOCAL SPECTRAL IMAGING
Edmond Kahn; Vejux Anne; Dominique Dumas; Frouin
Frederique; Gerard Lizard
108. EVALUATION OF ITAG MHC TETRAMERS FOR PREDICTION
OF RECURRENT OR PERSISTENT CYTOMEGALOVIRUS
INFECTION, DISEASE AND ASSOCIATED TRANSPLANTRELATED MORBIDITY OR MORTALITY IN ALLOGENEIC
STEM CELL TRANSPLANT RECIPIENTS: A PROSPECTIVE
MULTICENTER CLINICAL TRIAL
Jan W. Gratama; Michael Boeckh; Ryotaro Nakamura; Rik A.
Brooimans; Jan J. Cornelissen; John A. Zaia; Stephen J.
Forman; Karl Gaal; Gail H. Gasior; Linda A. Sullivan;
Christopher S. Boyce; Paula C. Southwick
109. MICROBEADS AS ARTIFICIAL APCS AND CELL BRIDGES
FOR T CELL CANCER THERAPY
Alfredo Prieto; Miguel Ángel Sánchez; Martin Villarroel;
David Diaz; Esperanza Perucha; Jorge Monserrat; Hugo
Barcenilla; Manuel Leonardo Acuña; Melchor Álvarez-Mon
110. STUDY OF CYTOKINE PRODUCTION BY NAÏVE,
EFFECTOR, MEMORY AND REGULATORY T CELLS BY 8
COLORS MULTIPARAMETRIC FLOW CYTOMETRY
Jorge Monserrat; Hugo Barcenilla; Angela HernáNdez;
Eduardo Reyes; Martin Villarroel; Miguel Ángel Sánchez;
David Diaz; Alfredo Prieto; Manuel Leonardo Acuña;
Melchor Álvarez-Mon
111. CD8+ T-CELL DYSFUNCTION IN AIDS: A CELLULAR
DEFECT OR AN ABSENCE OF CD4+ T CELLS HELP?
Patrick J. Autissier; Norman L. Letvin; Igor J. Koralnik; Joern
E. Schmitz
112. T CELLS HOMEOSTASIS IN CHILDREN WITH DOWN’S
SYNDROME
Erika Roat; Milena Nasi; Leonarda Troiano; Nicole Prada;
Marcello Pinti; Elisa Nemes; Enrico Lugli; Roberta Ferraresi;
Chiara Giovenzana; Luigi Ciacci; Ugo Consolo; Fiorella
Balli; Ornella Biagioni; Mauro Mariotti; Andrea Cossarizza
Wednesday, 24 May
115. NEXT-GENERATION CYTOMETRY: SPECTRAL
FINGERPRINTING
Bartlomiej Rajwa; Valeri Patsekin; J. Paul Robinson
116. MULTISPECTRAL IMAGING METHODS FOR MULTI-LABEL
MICROSCOPY
Richard Levenson; James Mansfield
117. MULTISPECTRAL FLUORESCENCE IMAGING OF SMALL
ANIMALS: DETECTION AND ANALYSIS METHODS USING
ACOUSTO-OPTIC FILTERING
Silas J. Leavesley; Valery P. Patsekin; Wamiq Ahmed;
Bartlomiej Rajwa; J. Paul Robinson
118. CONFOCAL MICROSCOPY SYSTEM
PERFORMANCE:SPECTROSCOPY
Robert Martin Zucker; Jeremy Lerner
43
Biotechnology ..................................... Room 302
08:00 - 09:30
Chairs: Patrick Daugherty and Bruce Edwards
119. THE USE OF MULTI-PARAMETER FLOW CYTOMETRY FOR
THE CHARACTERISATION AND MONITORING OF INSECT
CELL-BACULOVIRUS FERMENTATIONS IN A
MECHANICALLY-AGITATED BIOREACTOR
Christopher J. Hewitt; Bojan Isailovic; Ryan Hicks; Ian Taylor
120. DETERMINATION OF FLUORESCENCE IN SITU
HYBRIDIZATION POSITIVE EVENTS WITH AN IMAGING
FLOW CYTOMETER – FISH IN FLOW
Luchuan Liang; Philip Morrissey; Brian Hall; Thaddeus
George; David Basiji; William Ortyn; Keith Frost; Cathleen
Zimmerman; Richard Esposito; Richard Bauer; David J.
Perry
121. BEYOND ‘ONE-IN-A-BILLION´: RARE CELL SORTING IN
DIAGNOSTICS AND THERAPEUTIC DEVELOPMENT
Patrick S. Daugherty
122. A MULTIPLEX FUNCTIONAL SCREEN OF PHAGE DISPLAY
LIBRARIES USING FLOW CYTOMETRY
Loretta Yang; John P. Nolan
123.YEAST SURFACE DISPLAY SYSTEM FOR SCREENING
OPTIMAL SUBSTRATE FOR ANTHRAX LETHAL FACTOR
Weon Bae; Jian Hong Zhou; Steven Graves
124. MORE ACCURATE GENETIC DIAGNOSIS BY SINGLE COPY
PROBE QUANTITATIVE MICROSPHERE HYBRIDIZATION
Heather Newkirk; Peter K. Rogan; Mauricio Miralles; Joan
H.M. Knoll
Apoptosis ............................................ Room 303
08:00 - 09:30
Chairs: Frank Traganos and Jianping Gong
125. HYPOXIN-INDUCED MEMBRANE-BOUND APOPTOTIC
DNA PARTICLES: POTENTIAL MECHANISM OF FETAL DNA
IN MATERNAL PLASMA
Aaron Orozco; Cassandra Horne; Edwina Jane Popek; Joe
Leigh Simpson; Farideh Z. Bischoff; Dorothy E. Lewis
126. A NOVEL FLOW CYTOMETRY BASED METHOD TO
DETERMINE THE VIABILITY OF BETA CELLS USING THE
ZINC-BINDING DYE FLUOZIN-3 AND THE
MITOCHONDRIAL MEMBRANE POTENTIAL INDICATOR
TMRE
Sundararajan Jayaraman
127. RELATIONSHIP BETWEEN MITOCHONDRIAL
DEPOLARIZATION AND OTHER APOPTOTIC EVENTS
Sundararajan Jayaraman; Jesus Exposito; Carlos Salgado
128. FLOW CYTOMETRY LEADS A SHIFT OF PARADIGM IN THE
ETHIOPATHOGENIC ROLE OF T LYMPHOCYTE APOPTOSIS
IN MULTIPLE SCLEROSIS
Hugo Barcenilla; Alfredo Prieto; David Diaz; Jorge
Monserrat; Manuel Leonardo Acuña; Antonio GarcíaMerino; Melchor Álvarez-Mon
44
129. MOLECULAR ORDERING OF THE FUNCTION OF CASPASE
3 AND 6 BY SIMULTANEOUS MEASUREMENT OF THE
ACTIVITIES OF TWO CASPASES IN LIVING CELLS WITH A
NOVEL DUAL FRET INDICATOR PROBE
Xiaoli Wu; James Simone; Liusheng He
130. SIMULTANEOUS MEASUREMENT OF APOPTOSIS AND
NUCLEAR TRANSLOCATION EVENTS USING THE
IMAGESTREAM IMAGING FLOW CYTOMETER
Thaddeus George; Brian Hall; David Basiji; Cathleen
Zimmerman; Keith Frost; William Ortyn; David J. Perry;
Richard Esposito; Richard Bauer; Philip Morrissey
Cancer Biomarkers ............................. Room 304
08:00 - 09:30
Chairs: Alison Allan and Patrice Petit
131. BIOMARKER-BASED CERVICAL CANCER SCREENING
USING FLOW CYTOMETRY - A NOVEL APPROACH
Jian Ling; Urs Wiederkehr; Spring Cabiness; Kenneth R.
Shroyer; J. Paul Robinson
132. HER2/NEU HERCEPTIN BIOMARKER DEVELOPMENT FOR
THERANOSTIC MANAGEMENT OF BREAST CANCER
PATIENTS
Ed Luther; Alexey Glazyrin; William Geddie; James Eliason
133. THE IMPACT OF TRASTUZUMAB, OMNITARG, AND
CETUXIMAB ON BREAST CANCER CELL PROLIFERATION
DEPENDS ON EGFR/HER2-COEXPRESSION
Gero Brockhoff; Elisabeth Schmidt-Bruecken; Ferdinand
Hofstaedter; Simone Diermeier
134. ANTIGEN MAPPING IN MYELODYSPLASIA:
ABNORMALITIES OF CD34 AND CD117 EXPRESSION ARE
COMMON
Samue lJ. Pirruccello; KenHE Young; Patricia Aoun
135. MONITORING MOLECULAR TARGETED THERAPEUTICS IN
PERIPHERAL BLOOD SAMPLES FROM ACUTE LEUKEMIA
PATIENTS BY FLOW CYTOMETRY
Sue Chow; Frances Tong; David Hedley
136. ANALYSIS OF VARIATION IN RESULTS OF FLOW
CYTOMETRIC ZAP-70 EXPRESSION ON B AND T
LYMPHOCYTES IN A MULTICENTER STUDY
Jaco Kraan
Environmental, Marine and
Microbiology ....................................... Room 202
08:00 - 09:30
Chairs: Gerald Gregori and Susann Müller
137. SHORT-TERM VARIATION OF THE PHYTOPLANKTON
ASSEMBLAGE IN THE BAY OF MARSEILLE (FRANCE)
MONITORED BY IN SITU FLOW CYTOMETRY
Melilotus Thyssen; Michel Denis
138. PRELIMINARY INVESTIGATION OF SYNECHOCOCCUS
AND PROCHLOROCOCCUS OF SOUTH WESTERN
WESTERN AUSTRALIA
Harriet Paterson; Kathryn Heel-Miller
139. CHARACTERIZATION OF ACANTHAMOEB A–
MICROSPHERE ASSOCIATION BY MULTI-PARAMETER FLOW
CYTOMETRY AND CONFOCAL MICROSCOPY
Christopher J. Hewitt; Steve Smith
140. POPULATION DYNAMICS WITHIN A MICROBIAL
CONSORTIUM DURING GROWTH ON DIESEL FUEL IN
SALINE ENVIRONMENTS
Susann Müller; Sabine Kleinsteuber; Ingo Fetzer;Hauke
Harms
141. MONITORING OF ANTI-MICROBIAL PROPERTIES OF
SPICE EXTRACTS AGAINST FOOD SPOILAGE BACTERIA
USING FLOW CYTOMETRY
Mercedes Alonso-Gomez; Martin Gerard Wilkinson; Edel
Durack
142. INDIVIDUALS BEHAVE DIFFERENTLY– MULTI-PARAMETER
FLOW CYTOMETRY FOR MONITORING BACILLUS CEREUS
BATCH FERMENTATION PROCESSES
Christopher J. Hewitt; Godfrey L William; Nichola Helen
Foxall; Andrew Want; Colin Thomas
New Investigator/Student Symposium
Monday, 22 May
16:00 – 17:00
Room 200C
Co-chairs: Laura Teodori and Zosia Maciorowski
Exceptional Student Award Finalists
President’s Award for Excellence Finalists
337/P191. AFFORDABLE CELL ENUMERATION FOR HIV
STAGING
Xiao Li, Aurel Ymeti, Bjorn Lunter, Christian
Breukers, Arjan G.J. Tibbe, Leon Terstappen, Jan Greve
115. NEXT-GENERATION CYTOMETRY: SPECTRAL
FINGERPRINTING
Bartolomej Rajwa, Valeri Patsekin, J. Paul Robinson
34. NONINVASIVE FORWARD-SCATTERING SYSTEM FOR RAPID
DETECTION, CHARACTERIZATION, AND IDENTIFICATION
OF LISTERIA COLONIES: IMAGE-PROCESSING AND
ANALYSIS
Bulent Bayraktar, Padmapriya P Banada, J. Paul
Robinson, Arun K. Bhunia, E. Daniel Hirleman, Bartlomiej
Rajwa
39. CLUSTER AND PRINCIPAL COMPONENT ANALYSIS FOR
THE IDENTIFICATION OF COMPLEX PHENOTYPES
Enrico Lugli, Marcello Pinti, Leonarda Troiano, Milena Nasi,
J. Paul Robinson, Valery Patsekine, Caterina Durante,
Gianfranco Salvioli, Marina Cocchi, Andrea Cossarizza
225/P79. INTEGRATION OF FLOW CYTOMETRY WITH SINGLE
CELL IMAGING PERMITS QUANTIFICATION OF
HERPESVIRUS INFECTION
Laura Adang, Chris Parsons, Dean H. Kedes
74. A COMPLEX DIETARY SUPPLEMENT DRAMATICALLY
ABERRATIONS, 8-OHDG LEVELS AND ãH2AX FOCI IN MICE
EXPRESSING ELEVATED FREE RADICAL PROCESSES
Jennifer Lemon, C. David Rollo, Douglas R. Boreham
165/P117. CELL DEATH IN LEUKOCYTES AND THE USE OF
ANNEXIN-V: CALCIUM MATTERS!
Uriel Trahtemberg, Mizhir Atallah, Alon Krispin, Inna
Verbovetski, Dror Mevorach
126. A NOVEL FLOW CYTOMETRY BASED METHOD TO
DETERMINE THE VIABILITY OF BETA CELLS USING THE
ZINC-BINDING DYE FLUOZIN-3 AND THE
MITOCHONDRIAL MEMBRANE POTENTIAL INDICATOR
TMRE
Sundararajan Jayaraman
82. FLOW CYTOMETRIC ANALYSIS AND PURIFICATION OF
NEURAL AND NEURONAL CELL POPULATIONS DERIVED
FROM HUMAN EMBRYONIC STEM CELLS
Jan Pruszak, Kai-Christian Sonntag, Joris Van Arensbergen,
Takahito Yoshizaki, Moe Hein Aung, Rosario SanchezPernaute, Ole Isacson
31. A GENERAL TECHNIQUE FOR SEGMENTATION OF
Zachary Pinkus, Julie A. Theriot
ISAC Scholars
Dario Coletti, Postdoc, Italy
Tytus Bernas, Postdoc, Poland
Dominik Lenz, Postdoc, US
Lori Yang, Postdoc, US
Ryan Brinkman, Assistant Professor, Canada
Claudio Panzarella, PhD student, Italy
Reiner Schulte, PhD student, Germany
Laura Adang, PhD student, US
45
46
P141
P197
P10
P315
P286
P36
P179
P22
P222
P77
P91
P161
P9
Achuthanandam, Ram
Agustin, Ramses
Balazs, Margit Sr.
Bee, Wei-Lin Tiger
Biran, Israel
Chara Velarde, Luis E.
Congreve, Aileen
Conolly, Josh J.
Constantinou, Paul
Delaporte, Maryse M.
Grafton, Meggie
Haglund, Emily
Horváth, Viktor
306
P160
P249
P11
P140
Seale, Mary-Margaret
Sinnie, Ng
Soucek, Karel
Spidlen, Josef
286
382
159
P114
474
482
451
448
475
217
215
355
348
193
401
155
361
356
Iranpour Feridani, Amir H.
Jonasdottir, Oktavia
P350
Jonasdottir, Oktavia
P358
Kappelmayer, János
P327
Klabusay, Martin
P324
Klabusay, Martin
P351
Kotova, Natalia
P71
Leysi-Derilou,Younes
P69
Mazzini, Giuliano
P209
Mittag, Anja
P202
Nolan, Rhiannon L.
P45
Okada, Seiji
P268
Palyi-Krekk, Zsuzsanna P7
Prigozhina, Natalie
P215
Quinn, John
P210
157
325
170
368
223
237
307
287
343
158
439
419
184
Poster B Prog.
Primary Author
Secondary Authors
Capocasale, Renold J.; Quinn, John; Bugelski, Peter; Hrebien, Leonid; Kam, Moshe
Azimi, Behrad; Shih, Wenting; Price, Jeffrey
Treszl, Andrea; Rakosy, Zsuzsa; Bégány, Ágnes; Adany, Roza
Lenz, Dominik; Ling, Jian; Robinson, J. Paul
Haigh, Sarah; Zurgil, Naomi; Deutsch, Motti; Shirihai, Orian
Chevarria, Julio; Diaz, David; Prieto, Alfredo; Monserrat, Jorge; Barcenilla, Hugo;
Sanchez, Miguel A.; Acuna, Leonardo; Munoz, Norman; Alvarez-Mon, Melchor
Weir, Iona E.; Baguley, Bruce C.
Hedley, David; Wilson, Brian
McKenna, Patty; Berthe, Franck
Reece, Lisa M.; Lim, KwanSeop; Liu, Yi-Shao; Bashir, Rashid; Leary, James F.
Seale, Mary-Margaret; Zordan, Michael D.; Cooper, Christy L.; Reece, Lisa M.;
Huang, Jianjie; Prow, Tarl W.; Bergstrom, Donald Eugene; Leary, James F.;
Cancer Biology
Soucek, Karel; Svihalkova-Sindlerova, Lenka; Hofmanova, Jirina; Sova, Petr;
Kozubík, Alois
260
Flow Cytometry - Technical
Baldetorp, Bo
Leukemia/ Lymphoma
Olesen, Gitte; Thisted, Marianne; Just, Tom
Leukemia/ Lymphoma
Olesen, Gitte; Thisted, Marianne; Just, Tom
Hematopoesis and Stem Cell Sziráki Kiss, Valéria; Karászi, Éva
Hematopoesis and Stem Cell Koristek, Zdenek; Kohutova, Viera; Vinklarkova, Jaroslava; Mayer, Jiri
Leukemia/ Lymphoma
Sukova, Vera; Coupek, Petr
Other
Grawé, Jan
Cell Growth and Differentiatio Duchesne, Carl; Hains, Marie-Christine; Pineault, Nicolas; Garnier, Alain
Advanced Microscopy
Angelini, Marco; Beller, Thomas; Dei Tos, Angelo Paolo; Giangarè, Maria Chiara
Image Cytometry
Mosch, Birgit; Arendt, Thomas; Tárnok, Attila
Cell Function
Kimes, Nikole; Flippin, Jessica; Goldstein, Lawrence S.
Immunology/ Hematology
Goto, Yumi; Harada, Hideki; Suzu, Shinya
Cancer Biology
Barok, Márk; Tammi, Markku; Vereb, Gyorgy; Nagy, Péter; Szöllõsi, János
Digital Imaging-Software
Callaway, Scott; Mikic, Ivana; Hunter, Edward; Price, Jeffrey; McDonough, Patrick
Bioinformatics
Fisher, Paul W.; Capocasale, Renold J.; Achuthanandam, Ram; Kam, Moshe;
Bugelski, Peter; Hrebien, Leonid
Molecular Pharmacology
Haglund, Emily; Zordan, Michael D.; Cooper, Christy L.; Reece, Lisa M.; Huang, Jianjie;
Prow, Tarl W; Bergstrom, Donald Eugene; Leary, James F.
Cancer Biology
Phung, Anh D.; Bulinski, J. Chloe; Harper, Richart W.; Mc Manus, Michael T.; Eserich,
Jason P.
Bioinformatics
Gentleman, Robert; Haaland, Perry; Ochs, Michael F.; Schmitt, Charles; Smith,
Clayton; Treister, Adam S.; Brinkman, Ryan R.
High Throughput Analysis
Cell Death
Spectral Imaging
Aquatic Sciences
BioMEMs/microfabrication
Image Cytometry
Cancer Biology
Biomarkers for predicting ther
Cell Death
Category
Outstanding Poster Award Applicants
47
P21
P332
P144
P162
P338
P276
P284
P128
P52
P23
P164
P40
P176
Takao,Tania Tieko
Vejux, Anne M.
Vlkova, Marcela
Wang, Lili
Welzenbach, Karl
Wing Yin, Ho
Wittenbrink, Nicole
Wittenbrink, Nicole
Wu,Yang
Xie, Daxing
Xie, Daxing
Zhang, Jingli
Zhang, Ping
Zordan, Michael D
198
171
310
188
322
169
456
290
308
462
409
417
274
466
Poster B Prog.
P342
Primary Author
Secondary Authors
Cole, Kenneth; He, Hua-Jun; Hancock, Diane; Zong, Yaping
Krähenbühl, Stephan; Weitz-Schmidt, Gabriele
Dorlhiac - Llacer, Pedro Enrique; Velloso, Elvira; Munhoz, Marcos Antonio; Sales,
Maria Mirtes
Montange,Thomas; Kahn, Edmond; Lizard, Gérard
Klein, Anke; Schuchhardt, Johannes; Or-Guil, Michal
Klein, Anke; Schuchhardt, Johannes; Or-Guil, Michal
Campos, Samuel K.; Lopez, Gabriel P.; Ozbun, Michelle A.; Sklar, Larry A.;
Buranda,Tione
Cell Growth and Differentiatio Wu, Jianhong; Feng, Yongdong; Li, Xiaolan; Tao, Deding; Gong, Jianping
Cell Death
Feng, Yongdong; Zhang, Peng; Wu, Jianhong; Tao, Deding; Gong, Jianping
Pharmaceutical Applications Stevenson, David; Adaim, Aselle; Stanley, Roger; Skinner, Margot
Cell Function
Zuo, Hui; Chen, Wan-tao; Kakudo, Kennichi
High Throughput Analysis
Reece, Lisa M.; Leary, James F.
Cell Death
Immunology and AIDS
Proteomics
Inflammation
Category
Outstanding Poster Award Applicants (continued)
Poster Presentations
Exhibit Hall 400 B/C
Program abstract number followed by poster board number.
149/P1. MULTICOLOR FLOW CYTOMETRIC ERBB RECEPTOR
AND DNA QUANTIFICATION IN BREAST CANCER CELLS
Arabel Vollmann; Gero Brockhoff; Ferdinand Hofstaedter
150/P2. ESTABLISHMENT OF ORAL CANCER CELL LINES AND
IDENTIFICATION OF THEIR MOLECULAR PHENOTYPE
Wan-Tao Chen; Ronggen He; Xiaojian Zhou; Ping Zhang
151/P3. SCREENING AND IDENTIFICATION THE CISPLATINRESISTANCE RELATED GENES IN HUMAN ORAL
SQUAMOUS CELL CARCINOMA CELL LINE
Ping Zhang; Wan-Tao Chen; Xiaojian Zhou; Qin Xu;
Mingbin Zhang; Weiliu Qiu
152/P4. MULTIPLEXED FLUORESCENCE INSITU
HYBRIDIZATION OF ERBB RECEPTOR STATUS
Andrea Sassen; Simone Diermeier; Arabel Vollmann;
Stephan Schwarz; Gero Brockhoff
153/P5. ANALYSIS OF THE CYTOKINE PROFILE TH1/TH2
AGAINST THE HUMAN PAPILLOMAVIRUS TYPE 16 E7
ONCOPROTEIN IN PATIENTS WITH INVASIVE CERVICAL
CANCER RECEIVING RADIOTHERAPY
Felix G. Delgado; Alba L Combita; Maria A. Cespedes;
Alexander Rodriguez; Maria M. Bravo
154/P6. FLOW CYTOMETRY FOR IMMUNOPHENOTYPING
BREAST, OVARY AND COLON CANCER
Rafael Nunez; John Magenau; Sergio Zamorano; Antonella
Lostumbo; Divyesh Mehta
155/P7. THE ROLE OF CD44 IN THE MALIGNANT PHENOTYPE
OF A TRASTUZUMAB RESISTANT BREAST CANCER CELL
LINE
Zsuzsanna Palyi-Krekk; Márk Barok; Markku Tammi; Jorma
Isola; Gyorgy Vereb; Péter Nagy; János Szöllõsi
156/P8. COMPARATIVE EFFECTS OF RESVERATROL,
RESVERATROL ANALOGUES AND VINEATROL ON THE CELL
CYCLE OF COLON TUMORAL CELL LINES
Dominique Delmas; Anna-Kristina Marel; Gérard Lizard;
Norbert Latruffe
157/P9. DIFFERENCES IN CELL CYCLE REGULATION AFTER
PLATINUM DERIVATIVES TREATMENT IN SENSITIVE AND
CISPLATIN RESISTANT OVARIAN CANCER CELL LINES
Viktor Horváth; Karel Soucek; Lenka Svihalkova-Sindlerova;
Jirina Hofmanova; Petr Sova; Alois Kozubí K.
158/P10. WHOLE GENOME SCREEN OF PRIMARY
MELANOMAS BY ARRAY CGH
Margit Balazs; Andrea Treszl; Zsuzsa Rakosy; Ágnes
Bégány; Roza Adany
48
159/P11.TUBULIN TYROSINE LIGASE EXPRESSION
CORRESPONDS TO CHANGES IN THE TYROSINATION/
DETYROSINATION STATUS OF-TUBULIN IN PROSTATE
CANCER CELLS
Karel Soucek; Anh D. Phung; J. Chloe Bulinski; Richart W.
Harper; Michael T. McManus; Jason P. Eserich
160/P12. CD31 (PECAM-1) AND CD38 ANTIGENS ARE COLOCALIZED ON THE CELL SURFACE OF HL-60 HUMAN
MYELOBLASTIC CELL LINE
Olivier Herault; Nathalie Gallay; Michel Degenne; MarieThétèse Georget; Jorge Domenech; Christian Binet
161/P13. SELECTION OF AGONISTIC ANTIBODIES TO A
RECEPTOR TYROSINE KINASE USING FLOW CYTOMETRYBASED ASSAYS FOR DETECTION OF TOTAL TYROSINE
PHOSPHORYLATION AND RECEPTOR INTERNALIZATION
Paul Larsen; Siew Schleyer; John Kunich; David Bohmann;
Lynn Webster; Eddie Bautista; Jeff Hsu; Judith Abraham;
Masahisa Handa
162/P14. IDENTIFICATION OF NEUTRALIZING ANTIBODIES TO
A G PROTEIN-COUPLED RECEPTOR (GPCR) LIGAND USING
A FLOW CYTOMETRY-BASED INTRACELLULAR CALCIUM
FLUX ASSAY
Paul Larsen; Amita Patel; Elizabeth Pongo; David
Bohmann; Jody Brink; Tamlyn Neben; Marina Roell;
Rhonda Hansen; Steve Grimes; Masahisa Handa
163/P15. HUMAN GLIOBLASTOMA CELL LINES ARE RESISTANT
TO RADIATION-INDUCED APOPTOSIS EXPRESS HIGH
LEVEL OF BCL-2 AND METALLOTHIONEIN AND DO NOT
DEPLETE GLUTATHION
Maria Giovanna Valente; Claudio Panzarella; Dario Coletti;
Wolfgang Goehde; Burkhard Greve; Krzysztof Jamroziak;
Piotr Smolewski; Tadeusz Robak; Laura Teodori
164/P16. MULTIPARAMETER FLOW CYTOMETRIC ASSAYS FOR
THE STUDY OF APOPTOSIS
Carl Bortner; Maria Sifre; John Cidlowski
165/P17. CELL DEATH IN LEUKOCYTES AND THE USE OF
Uriel Trahtemberg; Mizhir Atallah; Alon Krispin; Inna
Verbovetski; Dror Mevorach
166/P18. REDUCTION OF SPONTANEOUS APOPTOSIS, BAK
AND BAX IN LARYNGEAL CARCINOMA
Gg Chen; Ac Vlantis; Ecw Chak; Hc Liu; Mcf Tong; Ca Van
Hasselt
167/P19. MEASUREMENT OF APOPTOSIS IN GROWING CELL
POPULATIONS
David Diaz; Alfredo Prieto; Hugo Barcenilla; Jorge
Monserrat; Luis Chara; Julio Chevarria; Miguel Ángel
Sánchez; Eduardo Reyes; Melchor Álvarez-Mon
168/P20. ABNORMAL FAS/FASL AND CASPASE-3-MEDIATED
APOPTOTIC SIGNALING PATHWAYS OF T LYMPHOCYTE
SUBSETS IN PATIENTS WITH SYSTEMIC LUPUS
ERYTHEMATOSUS
Lanlan Wang; Bei Cai; Xue Chen; Jie Chen; Weihua Feng;
Honggang Wei
169/P21. FLUOROCHROME-LABELED INHIBITORS OF
CASPASES (FLICA) STAIN CELLS WITH FRAGMENTED,
CONDENSED AND SWOLLEN NUCLEI DURING 7KETOCHOLESTEROL-INDUCED CELL DEATH
Anne Vejux; Thomas Montange; Edmond Kahn; Gérard
Lizard
170/P22. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES
IN PLANT CELLS DURING AUTOPHAGY
Josh J. Conolly; Iona E. Weir; Bruce C. Baguley
171/P23. THE INFLUENCE ON CELL CYCLE CHECKPOINTS
AND APOPTOSIS INDUCED BY DIFFERENT DOSAGES OF XRAY
Daxing Xie; Yongdong Feng; Peng Zhang; Jianhong Wu;
Deding Tao; Jianping Gong
172/P24. WRONG TIME AND WRONG PLACE OF CDK
ACTIVATION FOR G1 APOPTOSIS IN LEUKAEMIA CELL LINE
AND HUMAN PBL
Jianhong Wu; Yongdong Feng; Xiaolan Li; Daxing Xie;
Deding Tao; Junbo Hu; Jianping Gong
173/P25. MEMORY EFFECTS OF THE G1-PHASE CELL CYCLE
CHECKPOINTS
Yixin Tong; Daxing Xie; Deding Tao; Junbo Hu; Jianping
Gong
174/P26. CELL CYCLE SPECIFICITY OF APOPTOSIS IN
DIFFERENT COMBINATION TREATMENTS IN MOLT-4 CELL
LINE
Yongdong Feng; Chunzhao Yu; Deding Tao; Jianhong Wu;
Jianping Gong
175/P27. EFFECTS OF CAMPTOTHECIN AND CYTOSINE
ARABINOSIDE ON CELL CYCLE SPECIFICITY OF
APOPTOTIC CELLS DURING COMBINATION TREATMENTS
IN MOLT-4 CELL LINES IN VITRO
Deding Tao; Chunzhao Yu; Hui Xiao; Xiaolan Li; Jianhong
Wu; Jianping Gong
180/P32. APOPTOTIC RATE VS APOPTOTIC INDEX IN THE
QUANTIFICATION OF APOPTOTIC CELLS IN BOTH RAT
AND MOUSE CELL CULTURES
Julio Chevarria; Luis Chara; David Diaz; Alfredo Prieto;
Jorge Monserrat; Hugo Barcenilla; Miguel A. Sánchez;
Leonardo Acuña; Norman Muñoz; Melchor Alvarez De
Mom
181/P33. LATE APOPTOTIC CHANGES IN CHROMATIN
STRUCTURE AND DNA CONTENT DETECTED WITH
VYBRANT DYECYCLE STAINS
Jolene A. Bradford; William L. Godfrey
182/P34. SIX-COLOR CYTOMETRIC ANALYSIS OF FAS/FASL
REGULATION OF MURINE BASOPHILIC ERYTHROBLAST
HOMEOSTASIS
Renold J. Capocasale; Dorie Makropoulos; Amy Volk;
Jeffrey Arlen; John Quinn; Ram Achuthanandam; Peter
Bugelski
183/P35. CHARACTERIZATION OF AGONISTIC CD28
SIGNALING IN JURKAT AND H9 T CELLS
Mary A. Turner; Michael McDermott; Ashvin Dewan;
Ashley Martin; John Rodgers; Dorothy E. Lewis
184/P36. LOSS OF LINEAGE ANTIGENS IN RAT AND MOUSE
APOPTOTIC LYMPHOCYTES
Luis E. Chara Velarde; Julio Chevarria; David Diaz; Alfredo
Prieto; Jorge Monserrat; Hugo Barcenilla; Miguel A.
Sanchez; Leonardo Acuna; Norman Munoz; Melchor
Alvarez-Mon
185/P37. EFFECTS OF HEAT SHOCK PROTEIN 72 ON
NEUTROPHIL FUNCTION
Andrew Osterburg; Sandy Schwemberger; George F.
Babcock
186/P38. OXIDATIVE STRESS PRODUCE CHANGES IN DNA
TOPOLOGY OF HUMAN LEUKOCYTES
Andrey I. Gorevich Poletaev; Andrey Neustroev; Maria
Vladimirovna Savvateeva
176/P28. COMPARISON OF CALCEIN-AM AND ANNEXIN-V AS
EARLY VITAL MARKERS OF APOPTOSIS BY CYTOMETRY
Yongdong Feng; Jia Wang; Xiaolan Li; Hui Xiao; Jianping
Gong
187/P39. KINETICS AND EXPRESSION OF CD59 EXPRESSION
IN CHO AL CELLS ALTERED WITH PHOSPHOLIPASE C AND
RNAI
Carley Ross; Michael H Fox
177/P29. FAS EXPRESSION IN G1 PHASE WAS CORRELATED TO
CELL CYCLE SPECIFIC APOPTOSIS IN PHA STIMULATED
PBL CELLS
Jing Hu; Yongdong Feng; Daxing Xie; Deding Tao; Jianping
Gong
188/P40. IDENTIFICATION OF SMALL SUBPOPULATION OF
CELLS FROM HUMAN ANAPLASTIC THYROID CARCINOMA
Ping Zhang; Hui Zuo; Wan-Tao Chen; Kennichi Kakudo
178/P30. APOPTOSIS AND PROLIFERATION OF ORAL
MUCOSAL EPITHELIA CELLS AFFECTED BY THE
NUTRITIONAL STATUS
Xuelai Luo; Deding Tao; Chuanyong Yang; Junbo Hu;
Jianping Gong
179/P31. NOVEL VIOLET-EXCITED REAGENTS FOR DETECTION
OF VIABILITY AND VITALITY
Gayle Marie Buller; JoleneA. Bradford; Stephen Yue;
Jixiang Liu; William L. Godfrey
189/P41. ADHERENT CELL DISSOCIATION FOR FLOW
CYTOMETRIC PHOSPHOPROTEIN ANALYSIS
Piet Van Erp; Diana Olthuis; Lisa Green; Rita Bowers;
Haiyan Long; Philip Marder
190/P42. IMPROVED ANALYSIS OF INDIVIDUAL
MITOCHONDRIA BY HIGH SENSITIVITY FLOW CYTOMETRY
James P. Freyer; Claire Sanders; Stephanie Field; Robb
Habbersett
191/P43. MULTIPARAMETRIC FLOW CYTOMETRIC
EVALUATION OF CYTOTOXIC T LYMPHOCYTE
POPULATIONS
Julie G. Wilkinson; Sybil S. D’Costa; Enrique Rabellino
49
192/P44. STUDY OF CYTOKINE PRODUCTION BY FOXP3
EXPRESSING CELLS BY 8 COLORS MULTIPARAMETRIC
FLOW CYTOMETRY
Manuel Leonardo Acuña; Hugo Barcenilla; Jorge
Monserrat; Alfredo Prieto; David Diaz; Martin Villarroel;
Angela Hernández; Eduardo Reyes; Luis Chara; Julio
Chevarria; Melchor Álvarez De Mon
193/P45. DERIVATION AND CHARACTERIZATION OF HES
CELLS LINES WITH CRE-DEPENDENT RNAI OF KINESIN-1
SUBUNIT KLC1
Rhiannon L. Nolan; Nikole Kimes; Jessica Flippin;
Lawrence S. Goldstein
194/P47. RESTRICTION POINT DEFINITELY EXISTS IN NORMAL
LYMPHOCYTES AND IS BROKEN IN MOLT-4 LEUKEMIC
CELLS
Jichao Qin; Yongdong Feng; Xiaolan Li; Daxing Xie; Hui
Xiao; Deding Tao; Junbo Hu; Jianping Gong
195/P48. EXPRESSION OF CYCLINS AND CDKS IN NORMAL
PROLIFERATING BONE MARROW CELLS IN VIVO
Daxing Xie; Jing Yao; Deding Tao; Junbo Hu; Yongdong
Feng; Jianping Gong
196/P49. EXPRESSION OF CYCLINS IN HIGH-DENSITY
CULTURED CELLS AND IN VIVO GROWING CELLS
Daxing Xie; Deding Tao; Jing Yao; Junbo Hu; Jianhong Wu;
Xiaolan Li; Jianping Gong
197/P51. DYNAMIC BINDING OF PCG PROTEINS DURING
DROSOPHILA DEVELOPMENT
Jasper Grendel; Cornelia Fritsch; Gabriella Ficz; Rainer
Heintzmann; DonnaJ. Arndt-Jovin
198/P52. ANALYSIS OF UV INDUCED DNA DAMAGE
CHECKPOINT BY CYCLIN E/DNA MULTIPARAMETER FLOW
CYTOMETRY
Daxing Xie; Jianhong Wu; Yongdong Feng; Xiaolan Li;
Deding Tao; Jianping Gong
199/P53. STUDY ON CYCLIN B1 AND CYCLIN E GENE
EXPRESSION IN POST-SORTING CELLS BY QUANTITATIVE
REAL-TIME PCR TECHNIQUE
Hui Xiao; Daxing Xie; Xiaolan Li; Yongdong Feng; Deding
Tao; Jianping Gong
200/P54. INITIAL TIME OF APOPTOSIS WAS DEPENDENT ON
CELL CYCLE PROGRESSION IN HUMAN PBL AND
LEUKEMIA CELL LINES
Yongdong Feng; Jianhong Wu; Junbo Hu; Daxing Xie;
Xiaolan Feng; Jianping Gong
201/P55. FLOW CYTOMETRIC ANALYSIS OF CELL CYCLE
SPECIFIC DIFFERENTIATION OF HL-60 CELLS
Ruojian Wen; Daxing Xie; Peng Zhang; Deding Tao;
Jianping Gong
202/P56. CULTURE OF CELLS FROM PLACENTA AND BONE
MARROW IN BFGF CONTAINING MEDIUM GIVE RISE TO
FRIZZLED-9 AND SSEA-4 EXPRESSING MSC WITH MULTIPOTENTIAL DIFFERENTIATION CAPACITY
Hans-Jorg Buhring; Venkata Lokesh Battula; Andreas
Boehmler; Sabrina Treml; Petra Bareiss; Ingrid Albert; Sigrid
Hojak; Hans Kiefer; Lothar Just; Thomas Skutella
203/P57. MULTIPLEXED CHARACTERIZATION AND
MONITORING OF CULTURED ADULT MESENCHYMAL STEM
CELLS
Shayne Boucher; Kate Wagner; Ferenc Boldog
50
204/P58. CELL CYCLE ANALYSIS USING MICROPLATE
CYTOMETRY: A COMPARISON OF LASER AND DYE
COMBINATIONS
Tristan Cope; Christopher Lupton; Jolene A. Bradford; Jeff
Hung; Wayne P. Bowen
205/P59. IMPROVED METHODS FOR PLATELET ANALYSIS BY
FACS AND FOR STABILIZATION OF CD42B EXPRESSION
ON CORD BLOOD CULTURE-DERIVED PLATELETS
Lucie Boyer; Nicolas Pineault
206/P60. CELL CYCLE ANALYSIS IN LIVE CELLS USING NOVEL
VYBRANT & REG DYECYCLESTAINS WITH VIOLET, BLUE,
AND GREEN EXCITATION
Jolene A. Bradford; Pam Whitney; Timothy Huang; Patrick
Pinson; Ching-Ying Cheng; Stephen Yue; William L.
Godfrey
207/P61. EFFECT OF X-IRRADIATION ON EARLY GASTRULAS
IN NORMAL MICE, DNA-REPAIR DEFICIENT MICE AND
MICE DEFICIENT IN CELL CYCLE CONTROL
Sarah Baatout; Jasmine Buset; Mieke Neefs; Arlette
Michaux; Bernard Chatelain; Hanane Derradji; Paul
Jacquet
208/P62. PHENOTYPE AND FUNCTION OF CD56BRIGHT NK
CELLS GENERATED IN VITRO FROM HEMATOPOIETIC
PROGENITOR CELLS: IDENTIFICATION OF AN CD56+/
GRANZYME B-/PERFORIN- FUNCTIONALLY IMMATURE
HUMAN NK CELL SUBSET
Loris Zamai; Claudia Masoni; Laura Galeotti; Barbara
Canonico; Massimo Della Felice; Fulvio Palma; Stefano
Papa
209/P63. USE OF FLOW CYTOMETRY IN THE VALIDATION OF
HUMAN MULTIPOTENT ADULT PROGENITOR CELLS
Amy C. Raber; Mark Frey; Pina Patel; Emily Rebro; Robert
Perry; Robert Deans; Wouter Vant Hof
210/P64. LIMB DEFECTS INDUCED BY X-IRRADIATION IN
MOUSE FOETUSES
Hanane Derradji; Sofie Bekaert; Rafi Benotmane; Patrick
Van Oostveldt; Sarah Baatout
211/P65. COMPARISON OF CFSE AND PKH26 WITH
CELLVUETM CLARET, A NEW 675NM-EMITTING
PROLIFERATION DYE
Andrew Bantly; Brian D. Gray; Elizabeth Breslin; Katharine
A. Muirhead; Betsy M. Ohlsson-Wilhelm; Jonni Moore
212/P66. ISOLATION OF ENRICHED PROGENITOR CELLS IN
THE PLANARIAN SCHMIDTEA MEDITERRANEA BY FLOW
CYTOMETRY
Namson-Hawk Oh; James C. Jenkin; Wayne F. Green;
Alejandro Sánchez Alvarado
213/P67. SU6656 INDUCES POLYPLOIDIZATION IN
MEGAKARYOCYTIC AS WELL AS IN CERTAIN LYMPHOMA
CELL LINES
Carl Simard; Nathalie Dussault; Serge Côté; Sonia Néron
214/P68. HEMATOPOIETIC CELLS CULTURED UNDER MILD
HYPERTHERMIA UNDERGO AN ACCELERATED CELL
DIFFERENTIATION AND PROLIFERATION KINETICS
Jean Francois Boucher; Nicolas Pineault
215/P69. MATHEMATICAL MODEL OF IN VITRO
MEGAKARYOPOIESIS
Younes Leysi-Derilou; Carl Duchesne; Marie-Christine
Hains; Nicolas Pineault; Alain Garnier
216/P70. IMPROVED MULTIPARAMETER FLOW CYTOMETRIC
DNA CONTENT ANALYSIS IN NEUROBLASTOMA USING
DRAQ5
Katrien Swerts;Yves Benoit; Geneviève Laureys; Jan Philippé
229/P83. ELECTROLYTIC DISINFECTION OF ESCHERICHIA
COLI AND COLIFORM BACTERIA IN A BATCH CELL WITH
DSA ELECTRODES
BradleyJAY Hernlem
217/P71. THE FREQUENCY OF MICRONUCLEI IN HUMANS
MEASURED BY FLOW: IMPACT OF DIET
Natalia Kotova; Jan Grawé
230/P84. QUANTITATIVE CHARACTERIZATION OF PROTECTIVE
ANTIGEN BINDING TO HUMAN TARGET CELL TYPES
Alina Deshpande; Claire Sanders; Rebecca Hammon;
Steven Graves
218/P72. SPIRULINA AS FOOD SUPPLEMENT FOR LONG-TERM
MANNED SPACE MISSIONS : EFFECT ON CANCER CELLS
Sarah Baatout; Hanane Derradji; Sofie Bekaert
219/P73. SPACE MICROBIOLOGY : PHYSIOLOGICAL
BEHAVIOUR OF BACTERIA
Sarah Baatout; Larissa Hendrickx; Natalie Leys; Max
Mergeay
220/P74. SHORT-TERM VARIABILITY OF ULTRAPLANKTON IN
THE NORTHWESTERN MEDITERRANEAN IN LATE SUMMER
2004. EVIDENCE FOR PULSED MINERALISATION IN THE
WATER COLUMN
Michel Denis; Melilotus Thyssen; Gérald Grégori; Cindy
Tavernier
221/P75. FIRST FLOW-CYTOMETRIC DETERMINATION OF
PICOPHYTOPLANKTON ABUNDANCE IN THE HUDSON
BAY AND ADJACENT WATERS: UNEXPECTED DOMINANCE
BY PICOCYANOBACTERIA
Claude Belzile; Michel Gosselin; Joannie Ferland; Mélanie
Simard
222/P76. WITHDRAWN
223/P77. IMMUNOPHENOTYPING NEOPLASTIC HAEMOCYTES
OF MYA ARENARIA: A NEW APPROACH IN DIAGNOSTICS
OF MOLLUSC DISEASES
Maryse M. Delaporte; Patty McKenna; Franck Berthe
224/P78. THE FLOW CYTOMETRY OF BACILLUS CEREUS
ENDOSPORES: CHARACTERISING AND QUANTIFYING
DAMAGE AND GERMINATION RATE
Ultan Philip Cronin; Martin Gerard Wilkinson
225/P79. INTEGRATION OF FLOW CYTOMETRY WITH SINGLE
Laura A. Adang; Chris Parsons; Dean H. Kedes
226/P80. APPLICATION OF FLOW CYTOMETRY TO
INVESTIGATE THE EFFECTS OF CELL ATTENUATION
METHODS ON PERMEABILITY AND INTRACELLULAR
ENZYME RELEASE FROM LACTOCOCCUS LACTIS SUBSP.
LACTIS 303
Imelda A. Doolan; Martin Gerard Wilkinson
227/P81. PHYSIOLOGICAL STUDIES OF THE COPPER
RESISTANCE OF CUPRIAVIDUS METALLIDURANS BY FLOW
CYTOMETRY
Sébastien Van Aelst; Abdelmalek Bahri; Max Mergeay;
Françoise Van Vliet; Sarah Baatout
228/P82. FLOW CYTOMETRY TO ANALYZE BACTERIA MARKED
WITH FLUORESCENT PROTEINS
Nico Boon; Arie Marel
231/P85. A DISSOCIATION AND STAINING PROCEDURE FOR
PARAFFIN-EMBEDDED TISSUES ENABLING FLOW-SORTING
OF NORMAL STROMAL CELLS AND TUMOUR CELL
SUBPOPULATIONS FOR FURTHER MOLECULAR GENETIC
ANALYSIS
Willem E. Corver; Natalja Ter Haar; Freek Blanken; Anya
Milne; Johan Offerhaus; Tom Van Wezel; Hans Morreau;
Cees J. Cornelisse; Gert Jan Fleuren
232/P86. MUTANT SPECTRA FROM GAMMA RADIATION AND
EMS MEASURED BY MULTICOLOR FLOW CYTOMETRY
Stephen B. Keysar; Carley D. Ross; C.Tenley French;
Michael H. Fox
233/P87. THE ACE SYSTEM, A MAMMALIAN ARTIFICIAL
CHROMOSOME ENGINEERING TECHNOLOGY:
GENERATION OF A PLATFORM ACE HOST CHO CELL LINE
FOR HIGH-YIELD RECOMBINANT PROTEIN PRODUCTION
G. Neil Mac Donald; Sandra Babich; Anne-Rachel
Fontanilla; Alexisann Maxwell; Diane Monteith; Joan
Shellard; Sharon Sidhu; Malcolm Kennard; Harry C.
Ledebur
234/P88. VH GENE USAGE AND SOMATIC MUTATION
DISTRIBUTION CONSISTENT WITH ANTIGEN-DRIVEN
SELECTION IN BOTH ‘MUTATED’ AND ‘UNMUTATED’ CASES
OF B-CELL CHRONIC LYMPHATIC LEUKEMIA
Karen Hensen; Brigitte Maes; Rita Smets; An Broekmans;
Sabine Franke; Greet Bries; Veerle Peeters; Reinoud
Cartuyvels; Jean-Luc Rummens
235/P89. DETECTION OF CHROMOSOMAL ABNORMALITIES
BY INTERPHASE FLUORESCENCE IN SITU HYBRIDISATION
ON FLOW SORTED PLASMA CELLS
Karen Hensen; Hanne Jongen; Sabine Franke; Veerle
Peeters; Brigitte Maes; Jean-Luc Rummens
236/P90. A SEMI DOUBLE EMULSION PROCESS FOR
RESERVOIR-TYPE MICROCAPSULES GENERATION
Sang-Youp Lee; Connie Snider; Kinam Park; J. Paul
Robinson
237/P91. MICROFABRICATION AND CONSTRUCTION OF A
BIOMEMS MICROFLUIDIC CELL SORTER
Meggie Grafton; Lisa M. Reece; Kwanseop Lim; Yi-Shao
Liu; Rashid Bashir; James F. Leary
238/P92. INTEGRATION OF MULTIPLE HIGH-SPEED DROPLET
CELL SORTERS INTO A STANDARD BIOSAFETY CABINET IN
A MANNER THAT PROVIDES BSL-2 CONTAINMENT
Gary Durack; Paul Weiss
239/P93. STERILE AND DISPOSABLE FLUIDIC SUB-SYSTEM
SUITABLE FOR CLINICAL HIGH SPEED FLUORESCENT
ACTIVATED CELL SORTING
Sach Jayasinghe; Joshua Wunderlich; Angel McKee;
Heather Newkirk; Linheng Li; Karen Staehling-Hampton;
Steve Pope; Jeffrey S. Haug
51
240/P94. REMOTE-CONTROLLED BIOHAZARD CELL SORTING
Kenneth Ketman; Natasha Barteneva
241/P95. LARGE PARTICLE SORTING WITHOUT A NOZZLE ON
THE BD FACS ARIA
David Houck
242/P96. LARGE PARTICLE FLOW CYTOMETRY FOR STUDY OF
CELL CLUSTERS
Rock Pulak; Bo Wang; Julia Thompson; Rico Bongaarts
243/P97. OPTIONS FOR A HIGH-SPEED PHOTODAMAGE CELL
SELECTION USING GOLD NANOPARTICLES AND PULSED
LASER IRRADIATION
Florian Levold; Sebastian Ziewer; Franziska Winter;
Gereon Huettmann; Johannes Gerdes; Elmar Endl
244/P98. IMPLEMENTATION OF A FREQUENCY BASED
METHOD FOR SORTING IN FLOW CYTOMETRY USING XML
Geoffrey W. Osborne; Geoffery Ericksson
245/P99. ACOUSTIC TECHNIQUE FOR ULTRASONIC PARTICLE
CONCENTRATION FOR UNIQUE FLOW CYTOMETRIC
ANALYSIS
Gregory Goddard; John C. Martin; Steven W. Graves;
Michael D. Ward; Gregory Kaduchak
246/P100. ON-CHIP NON-INVASIVE AND LABEL-FREE CELL
DISCRIMINATION BY IMPEDANCE SPECTROSCOPY
Marco Di Berardino; Grit Schade; Adrian Huwiler; Xavier
Houriet;Thomas Hessler
247/P101. A NOVEL FLAT-TOP BEAM SHAPER AND ITS
APPLICATION TO FLOW CYTOMETRY
Yong Q. Chen
248/P102. ANALYSIS OF HOECHST SIDE POPULATION CELLS
IN MOUSE BONE MARROW USING LOW-POWER UV
SOURCES
William G. Telford; Ella G. Frolova; Raquel Cabana; Richard
A. Thomas; Awtar Krishan
249/P103. MEASURING SIZE USING FORWARD ANGLE LIGHT
SCATTER IS NOT STRAIGHT FORWARD
Murugesan Venkatapathi; Gérald Grégori; E. Dan Hirleman;
J. Paul Robinson
250/P104. IMPROVED FORWARD SCATTER DETECTOR FOR
FLOW CYTOMETRIC CHARACTERIZATION OF SMALL
PARTICLES
Timothy Petersen; C. Brent Harrison; Jarred Swalwell; Ger
Van Den Engh
251/P105. LIGHT SCATTERING: STATE OF THE ART AND
FUTURE IN IDENTIFICATION AND CHARACTERIZATION OF
CELLS
Valeri P. Maltsev
252/P106. COMPUTER-SUPPORTED QUALITY CONTROL IN
DNA FLOWCYTOMETRY ANALYSIS
Nick Van Rodijnen; Eric Postma; Sjack Hoop; Mathy Leers;
Marius Nap
253/P107. AUTOMATED FLOW CYTOMETRY FOR STUDYING
TIME DEPENDENT CELL PHENOTYPES
Ann Hansgate; Alan Gilbert; Greg Sitton; Friedrich Srienc
52
254/P108. INFLUENCE OF FLOWCYTOMETERS AND
ACQUISITION/ANALYSIS SOFTWARES ON DETERMINATION
OF LYMPHOCYTE SUBSETS IN HIV INFECTION
Deshratn Asthana; Margarita Ashman; Naresh Sachdeva;
Leonardo Davilla; Gwendolyn B. Scott; Charles Mitchell;
Luis Cintron; Jose Moreno; Mobeen Rathore; Isaac Delke
255/P109. NOVEL CALIBRATION METHOD FOR FLOW
CYTOMETRIC FLUORESCENCE RESONANCE ENERGY
TRANSFER MEASUREMENTS BETWEEN VISIBLE
FLUORESCENT PROTEINS
Peter Nagy; László Bene; Manuela Braun; Christof Antz;
Jacques Paysan; Gyorgy Vereb; János SzöllõsSi
256/P110. QUANTITATIVE AND STATISTICAL COMPARISON OF
UNIVARIATE FLOW CYTOMETRY DATASTETS USING
HISTOGRAM SIMILARITY MEASURES
Tytus Bernas; Elikplimi Kwaku Asem; J. Paul Robinson;
Bartlomiej Rajwa
257/P111. WHEN THE LYMPHOSUM DOES NOT ADD UP
Michèle Bergeron; Tao Ding; Nadia Soucy; Naomi Lobo;
Francis Mandy
258/P112. EVALUATION OF MOUSE BONE MARROW BY FLOW
CYTOMETRY AFTER IN VIVO DOSING WITH
ERYTHROPOIETIN, GRANULOCYTE-COLONY STIMULATING
FACTOR OR 5-FLUOROURACIL
Cindy X. Zhang; David McFarland; Melanie Quinlan; Jie
Ding; Terry Sellers; Daniela Ennulat; Padma Kumar
Narayanan; Heath Thomas
259/P113. PACIFIC ORANGE™ DYE FOR USE IN
POLYCHROMATIC FLOW CYTOMETRY EXPERIMENTS
USING VIOLET DIODE LASER EXCITATION
Gayle Marie Buller; Stephen Yue; Jixiang Liu; William L.
Godfrey
260/P114. MULTICOLOUR FLOW CYTOMETRY TO QUANTIFY
INTERNALIZATION OF MONOCLONAL ANTIBODIES. A
NOVEL METHOD CONFIRMED BY CONFOCAL
MICROSCOPY
Amir H. Iranpour Feridani; Bo Baldetorp
261/P115. INTRODUCTION OF 7-LASER LSRII AND 5-LASER
FACSARIA
David Dombkowski; Larry Duckett; David Matsuyama;
Stephen Ziganti; Frederic Preffer
262/P116. FLOW CYTOMETRIC DETECTION OF ERYTHROCYTE
ZINC PROTOPORPHYRIN IN IRON DEFICIENT PATIENTS
Jörg Neukammer; Benedikt Krämer; Andreas Kummrow;
Sandra Schädel; Silke Heller; C. Thomas Nebe
263/P117. DEVELOPMENT OF MICROFLUIDIC STRUCTURES
FOR HIGH THROUGPUT FLOW CYTOMETRIC
CHARACTERIZATION OF BLOOD CELLS
Jörg Neukammer; Janko Theisen; Kerstin Brattke; Andreas
Kummrow; Thilo Guschauski; Martin Schmidt
264/P118. GATING METHODS FOR FLOWCYTOMETRY IN
MULTI-DIMENSION SPACE
Danhua Zhao
265/P119. GEOMETRIC ASPECTS OF CELL MEMBRANE
MEASUREMENTS AND INSTRUMENT DESIGN
Gordon W. Wiegand
266/P120. RESOLUTION REQUIREMENTS FOR THE
DIGITIZATION OF FLOW CYTOMETRY DATA
James C. S. Wood
267/P121. A 16-CHANNEL AVALANCHE PHOTODIODE
DETECTOR ARRAY FOR VISIBLE AND NEAR-INFRARED
FLOW CYTOMETRY
William G. Lawrence; Christopher Stapels; Richard Farrell;
Joseph Tario; Edward Podniesinski; Paul Wallace; James
Christian
268/P122. USE OF VIOLET-EMITTING AMINE REACTIVE DYE
COMPARED TO ETHIDIUM MONOAZIDE (EMA) FOR LIVEDEAD DETERMINATION IN INTRACELLULAR STAINING
ASSAYS
Martin Bigos; Ck Poon; Elizabeth Sinclair
269/P123. VALIDATION OF POLYCHROMATIC STAINING
PANELS TO DETECT T CELL SUBSET CYTOKINE RESPONSES
ON THREE FLOW CYTOMETER PLATFORMS
Bridget E. McLaughlin; Nicole Baumgarth; Martin Bigos;
Stephen C. De Rosa; John D. Altman; Mario Roederer;
Douglas Nixon; Janet Ottinger; Judy Li; Laurel A. Beckett;
David M. Asmuth
270/P124. OPTICAL FILTERS IN PRACTICE
Gouzel Tokmoulina
271/P125. QUANTITATION OF FLUORESCENCE
CONCENTRATION AND FLUORESCENCE SURFACE
DENSITY USING THE BECKMAN COULTER® CELL LAB
QUANTA™ SERIES FLOW CYTOMETERS
Michael Thomas; Raquel Cabana; Richard Thomas
272/P126. INDO-US CYTOMETRY WORKSHOPS
Awtar Krishan; L. Scott Cram
273/P127. FLOW CYTOMETRY COURSES FOR AFRICA
Claudio Vallan; Jennifer A. Wilshire; Adam Treister
274/P128. CALIBRATION OF QUANTUM DOTS AS PROBES OF
MOLECULAR ASSEMBLIES ON BEADS AND CELLS IN FLOW
CYTOMETRY AND MICROSCOPY
Yang Wu; Samuel K. Campos; Gabriel P. Lopez; Michelle A.
Ozbun; Larry A. Sklar; Tione Buranda
275/P129. IMMUNOFLUORESCENCE STANDARDIZATION TO
MEASURE THE NUMBER OF ANTIBODIES BOUND PER CELL
Doug Redelman
276/P130. INSTRUMENT CALIBRATION AND INTENSITY
MEASUREMENTS IN WIDE-FIELD AND CONFOCAL
FLUORESCENCE MICROSCOPY
Michael A. Model; James L. Blank
277/P131. A NEW SENSITIVITY TEST FOR BD FACSCANTO™
FLOW CYTOMETERS
James E. Bishop; Robert A. Hoffman; Mahrukh A. Huseni;
Hemangini Shah
278/P132. A FLEXIBLE AND EXTENSIBLE ARCHITECTURE FOR
VISUAL WORKFLOW DEVELOPMENT
Jaeick Oh
279/P133. PROPOSAL OF STANDARDS FOR COMPARING THE
PERFORMANCE AND SUITABILITY OF MULTIPLE
CYTOMETRY TECHNOLOGIES
Mel Henriksen
280/P134. INSTRUMENT CALIBRATION FOR DETERMINATION
OF RELATIVE FLUORESCENCE INTENSITY ON
POLYCHROMATIC FLOW CYTOMETERS
Constance Porretta; Judd Shellito; Steve Nelson; Ping
Zhang
281/P135. CONCEPT FOR THE TRACEABILITY OF
FLUORESCENCE (BEADS) IN FLOW CYTOMETRY:
EXPLOITING SATURATION AND MICROSCOPIC SINGLE
MOLECULE BLEACHING
Jörg Neukammer; Carsten Gohlke; Benedikt Kraemer;
Martin Roos
282/P136. CRITICAL ANALYSIS OF FLOW CYTOMETER
LINEARITY AND METHODS USED TO ASSESS IT
Robert A. Hoffman
283/P137. VARIABILITY OF OPTICAL FILTER SIGNAL TO NOISE
RATIOS IN THE RED EMISSION SPECTRUM FOR FLOW
CYTOMETRY
Lindsey Laycock; Gayle Thornbury; Gary De Jong
284/P128. EFFECTS OF INTRINSIC INTER-INSTRUMENT
VARIATION ON QUANTITATIVE FLOW CYTOMETRIC
CALCULATIONS
Ryan Duggan; David Leclerc
285/P139. DEVELOPMENT OF AN INTERSOCIETY
LABORATORY FLOW & IMAGING DATA EXCHANGE
SPECIFICATION AND/OR STANDARD
Robert Cary Leif
286/P140. PROPOSED GATING STANDARD FOR FLOW
CYTOMETRY
Josef Spidlen; Robert Gentleman; Perry Haaland; Michael
F. Ochs; Charles Schmitt; Clayton Smith; Adam S. Treister;
Ryan R. Brinkman
287/P141. EVALUATION OF THE EFFECTS OF ERYTHROPOIETIN
ON ERYTHROID PRECURSOR POPULATIONS IN MURINE
BONE MARROW USING A PATTERN RECOGNITION
APPROACH
Ram Achuthanandam; Renold J. Capocasale; John Quinn;
Peter Bugelski; Leonid Hrebien; Moshe Kam
288/P142. FULL AUTOMATION OF FLUORESCENCE
COMPENSATION AND EXTRACTION OF ADDITIONAL
INFORMATION OF VALUE FOR IMPROVING MULTI-COLOR
FACS ANALYSIS
David Parks; Wayne A. Moore
289/P143. APPLICATION OF TEMPORAL TEXTURE FEATURES
TO AUTOMATED ANALYSIS OF PROTEIN SUBCELLULAR
LOCATIONS IN TIME SERIES FLUORESCENCE MICROSCOPE
IMAGES
Yanhua Hu; Jesus Carmona; Theodore Scott Nowicki;
Robert F. Murphy
290/P144. COMPARISON OF MODEL PROTEIN
QUANTIFICATION USING FORWARD-PHASE PROTEIN
MICROARRAYS AND SUSPENSION ARRAYS
Lili Wang; Kenneth Cole; Hua-Jun He; Diane Hancock;
Yaping Zong
53
291/P145. INTEGRATION OF FLOW CYTOMETRY
TECHNOLOGY INTO A MASS SPECTROMETRY BASED
PROTEOMICS PLATFORM
Katherine McKinnon; Christine Evangelista; Elizabeth
Joseloff; Sudeepta Aggarwal; Tao He; Anne Deslattes
Mays; Paul Moore; Charles Birse; Steve Ruben
292/P146. EFFICIENT DELIVERY OF siRNA INTO DIVERSE CELL
TYPES WITH LOW TOXICITY VIA LASER-MEDIATED
OPTOINJECTION
Kate Rhodes; Imran Clark; Michelle Zatcoff; Trisha
Eustaquio; Kwame Hoyte; Manfred Koller
293/P147. ULTRASENSITIVE SINGLE-CELL AND POPULATIONLEVEL ANALYSES OF PROTEIN EXPRESSION IN
DEINOCOCCUS RADIODURANS BY CAPILLARY
ELECTROPHORESIS WITH LASER-INDUCED
FLUORESCENCE
Emily Turner; Norm Dovichi
294/P148. DATA QUALITY ASSESSMENT IN FLOW CYTOMETRY
EXPERIMENT
Nolwenn Le Meur; Robert Gentleman; Maura Gasparetto;
Clayton Smith; Ryan R. Brinkman
295/P149. PROTEOMIC STRATEGIES TO ANALYZE CELL-FREE
FRACTIONS FROM ACTIVATED PERIPHERAL BLOOD
MONONUCLEAR CULTURES
Sybil S. D’Costa; Julie G. Wilkinson; Enrique Rabellino
296/P150. IDENTIFICATION OF IMMUNE RESPONSE
SIGNATURES UTILIZING INTEGRATED CYTOMIC AND
PROTEOMIC TECHNIQUES
Sybil S. D’Costa; Julie G. Wilkinson; Enrique Rabellino
297/P151. CANCER RELATED CANDIDATE GENES SELECTION
AND MANUFACTURE THE FUNCTIONAL OLIGO
MICROARRAY OF ORAL SQUAMOUS CELL CARCINORMA
Liqiong Duan; Wan-Tao Chen; Ping Zhang; Xiaozhi Lu;
Ming Yan; Yan Lu; Jie He; Zhiyuan Zhang
298/P152. MICROARRAY-BASED GENOTYPING IN LARGE
POPULATIONS
David Galbraith; Jeremy Edwards; Ambika Gaikwad;
Jaroslav Janda; Stefan Schwab; Bin Liu; Hei Leung
299/P153. COMPARATIVE GENOMIC HYBRIDIZATION WITH IN
SITU SYNTHESIZED 60MER OLIGONUCLEOTIDE CGH
ARRAYS
Michael T. Barrett; Amir Ben-Dor; Anya Tsalenko; Doron
Lipson; Alicia Scheffer; Paige Anderson; Peter Tsang; Bo
Curry; Nick Sampas; Zohar Yakhini; Laurakay Bruhn
300/P154. DIFFERENTIAL GENE EXPRESSION IN HEPATOCYTE
SUBPOPULATIONS SORTED FROM ACETAMINOPHENTREATED MICE AND A COMPARISON OF 1 VS 3 DROP
SORTING
Collin C. White; Michael Dabrowski; Carolina Fernandez;
Richard Beyer; Theo Bammler; Terrance Kavanagh
301/P155. WITHDRAWN
302/P156. MULTI-ASSAY FOR DIFFERENTIATION OF INGESTED/
NON-INGESTED YEAST POPULATIONS AND
SIMULTANEOUS ASSESSMENT OF THE VIABILITY
Hans H. Bäumler; Verena Staedtke; Maria Fischer
54
303/P157. ENHANCED CYTOMETRIC BEAD ARRAY ASSAYS:
IMPROVING SENSITIVITY AND DECREASING ASSAY TIME
WITH A NOVEL AUTOMATED FLUIDICS APPROACH
Sujata Iyer; Julia Ember; Brandon Bowman; Rich M.
Ozanich; Cindy Bruckner-Lea; Brian Dockendorff
304/P158. EFFECT OF MICROSPHERE-BOUND SITE DENSITY
ON THE MEASURED AFFINITY OF AN INTERACTION
PARTNER
Marie Anne Iannone; Thomas G. Consler
305/P159. AUTOMATION OF TISSUE MICROARRAY ANALYSIS –
A NEW PARADIGM
Elena Holden; Alexey Glazyrin; Ed Luther
306/P160. DESIGN AND CONSTRUCTION OF
MULTIFUNCTIONAL PARAMAGNETIC NANOPARTICLES
Mary-Margaret Seale; Emily Haglund; Michael D. Zordan;
Christy L. Cooper; Lisa M. Reece; Jianjie Huang; Tarl W.
Prow; Donald Eugene Bergstrom; James F. Leary
307/P161. TARGETED, MULTIFUNCTIONAL QUANTUM DOT
NANOPARTICLES FOR EX VIVO DIAGNOSTICS
Emily Haglund; Mary-Margaret Seale; Michael D. Zordan;
Christy L Cooper; Lisa M. Reece; Jianjie Huang; Tarl W.
Prow; Donald Eugene Bergstrom; James F. Leary
308/P162. SIMULTANEOUS ASSESSMENT OF THREE IMMUNOPHARMACODYNAMIC PROPERTIES OF FUNCTIONALLY
DISTINCT LFA-1 ANTAGONISTS IN WHOLE BLOOD
Karl Welzenbach; Stephan Krähenbühl; Gabriele WeitzSchmidt
309/P163. SILICA - RED CELL ADSORPTIVE INTERACTION BY
LIGHT SCATTERING MEASUREMENTS
Igor I. Gerashchenko; BogdanI. Gerashchenko; Vasyl F.
Gorchev
310/P164. MODULATION OF TOXICITY OF CYTOKINES
SECRETED FROM THE HUMAN MONOCYTIC THP-1 CELLS
TOWARD SH-SY5Y NEUROBLASTOMA CELLS BY SELECTED
FLAVONOIDS AND THEIR GLUCURONIDES
Jingli Zhang; David Stevenson; Aselle Adaim; Roger
Stanley; Margot Skinner
311/P165. SINGLE CELL SORTING IN THE DESIGN OF AURORA
ASSAYS: SELECTION OF CELL POOLS EXPRESSING BETALACTAMASE UNDER TWO DIFFERENT PROMOTERS
Michael Cunningham; Marianna Kapitskaya; Bohumil
Bednar; Stefanie Kane; Konstantin Petrukhin
312/P166. GABA-B CELL LINE DEVELOPMENT
Christopher Donahue; Mei Cui; Fu-Zon Chung
313/P167. GABA-B AGONIST, POSITIVE ALLOSTERIC
MODULATOR AND ANTAGONIST ASSAY DEVELOPMENT
Christopher Donahue; Nicole Bart; Mei Cui; Brad Evans;
Justin Van Duine; Aaron Clark; Fu-Zon Chung
314/P168. LIMITS OF MINIATURIZATION IN HIGH-CONTENT
ANALYSIS: HOW BETTER DATA EXTRACTION CAN REDUCE
CELL NUMBER REQUIREMENT
Ilya Ravkin
315/P169. RAPID NON-INVASIVE ASSAYS FOR CELL GROWTH
AND INHIBITORY EFFECTS OF COMPOUNDS ON PRIMARY
CULTURES OF HUMAN UNLABELED CELLS
Marc Moeremans; Kris Ver Donck; Bieke Govaerts; Luc
Bols; Leen Geuens; Johan Geysen
316/P170. A NEW HIGH THROUGHPUT PROTEOMIC
APPROACH FOR RAID PROTEIN INTERACTION NETWORK
IDENTIFICATION USING TWO HYBRID SYSTEMS AND CELL
SORTING
Weon Bae; Jian Hong Zhou; Hong Cai
317/P171. USE OF FLOW CYTOMETRY TO SELECT LEAD
THERAPEUTIC ANTIBODY CANDIDATES
Masahisa Handa
318/P172. APPLICATION OF MICROPLATE CYTOMETRY TO
HIGH CONTENT SCREENING IN ONCOLOGY RESEARCH
Wayne P. Bowen
319/P173. CELL-BASED IMAGING TOOLS FOR HIGH CONTENT
SCREENING
Oleg Lapets; Everett Ramer; Richik N. Ghosh; Jeffrey R.
Haskins
320/P174. AUTOMATED MICROINJECTION SYSTEM FOR
SUSPENDED AND ADHERENT CELLS
Ian Welsford
321/P175. HIGH-THROUGHPUT SCANNING CYTOMETRY WITH
LASER OPTO-INJECTION OF MACROMOLECULES INTO
SELECTED CELLS (WITH LASER-MEDIATED ELIMINATION
OF UNDESIRED CELLS) FOR SUBSEQUENT GENE ARRAY
ANALYSES
James F. Leary; Peter Szaniszlo
322/P176. LASER OPTOINJECTION OF THERAPEUTIC
NANOPARTICLES INTO OSTEOSARCOMA CELLS
Michael D .Zordan; Lisa M. Reece; James F. Leary
323/P177. AUTOMATING LARGE SCALE, REPETITIVE, CLINICAL
FLOW CYTOMETRY ANALYSIS WITH THE CUSTOMIZED
PROTOCOL ENGINE FLOWDX
Adam Treister
324/P178. USING THE POLYVARIATE PLOT TOOL
Adam Treister; Maciej Simm
325/P179. SCREENING FOR PLANT BIOCATALYSTS USING
REACTIVITY BASED PROBES
Aileen Congreve
326/P180. HIGH THROUGHPUT METHOD FOR THE
IDENTIFICATION OF IN VIVO PROTEIN INTERACTIONS
AND MODIFICATIONS DURING D. MELANOGASTER
EMBRYOGENESIS
Joshua Wunderlich; Erika Geisbrecht; Kiranmai
Kocherlakota; David Ash; Mei-Hui Chen; Selene Swanson;
Laurence Florens; Michael Washburn; Susan Abmayr;
Jeffrey Haug
327/P181. EVALUATION OF A SINGLE AUTOMATED PLATFORM
FOR THE STANDARDIZATION OF FUNCTIONAL CELL
BASED ASSAYS IN PLATES
Julie G. Wilkinson; Aaron Globerman; Carlos Luis Aparicio;
Sybil S. D’Costa; Cecilia Smith; Enrique Rabellino
328/P182. WITHDRAWN
329/P183. THE PLACE AND USE OF AN IMAGE SCANNING
CYTOMETER IN A FLOW CYTOMETRY CORE
Gregory H. Veltri
330/P184. LASER SCANNING ANALYSIS OF CHROMATICALLY
LABELED TISSUE SECTIONS
Ed Luther; Lori Earls; William Geddie
331/P185. DIFFUSION PROPERTIES OF LIPOPHILIC DYES
WITHIN AND BETWEEN ADJACENT CELL MEMBRANES
Bernd Fritzsch; Katharine A. Muirhead; Brian D. Gray; Feng
Feng; Betsy Ohlsson-Wilhelm
332/P186. IMAGING CYTOMETRY METHODS FOR
CHARACTERIZING INTERNALIZATION
Gary Elliott
333/P187. CELL ORIENTING MICROSTRUCTURES FOR IMAGE
CYTOMETRY
Tycho M. Scholtens; Frederik Schreuder; Arjan G.J. Tibbe;
Leon W.W.M. Terstappen; Jan Greve
334/P188. A FAST IMAGING CYTOMETER
Frederik Schreuder; Tycho M. Scholtens; Sjoerd T. Ligthart;
Arjan G.J. Tibbe; Leon W.W.M. Terstappen; Jan Greve
335/P189. POINT OF CARE INSTRUMENTATION FOR
AFFORDABLE HIV MONITORING
Aurel Ymeti; Xiao Li; Bjorn Lunter; Christian Breukers; Arjan
G.J. Tibbe; Leon W.M.M. Terstappen; Jan Greve
336/P190. CELL POPULATION CLASSIFICATION BY FLOW
IMAGING ANALYSIS
Peter Oma
337/P191. AFFORDABLE CELL ENUMERATION FOR HIV
STAGING
Xiao Li; Aurel Ymeti; Bjorn Lunter; Christian Breukers; Arjan
G.J. Tibbe; Leon W.M.M. Terstappen; Jan Greve
338/P192. AUTOMATED FOUR COLOUR CD4/CD8 ANALYSIS
OF LEUKOCYTES BY SCANNING FLUORESCENCE
MICROSCOPY USING QUANTUMDOTS
Jozsef Bocsi; Anja Mittag; Viktor Sebestyen Varga; Bela
Molnar; Zsolt Tulassay; Ulrich Sack; Dominik Lenz; Attila
Tárnok
339/P193. AUTOMATED CLASSIFICATION OF APOPTOSIS AND
ARTIFACT REJECTION OF TUNEL POSITIVE CELLS
Brian Hall; Thaddeus George; David Basiji; Keith Frost;
Cathleen Zimmerman; William Ortyn; Philip Morrissey;
DavidJ Perry; Richard Esposito; Richard Bauer
340/P194. THE FRACTAL DIMENSION OF CHROMATIN IN
ROUTINE CYTOLOGY - A COMPARISON OF DIFFERENT
METHODS
Konradin Metze; Randall Luis Adam; Rosana C. Silva;
Neucimar Leite; Irene Lorand-Metze
341/P195. COMPARISON OF DIFFERENT KINDS OF ENTROPY
FOR THE CHARACTERIZATION OF NUCLEOLAR
ORGANIZER REGIONS ( AGNORS)
Konradin Metze; Randall Luis Adam; Ana Claudia S. Piaza;
Adilson A. Piaza; Neucimar J. Leite
342/P196. MULTIPLE DEMANDS—SINGLE SOLUTION:
BRINGING TOGETHER THE VARIOUS DIMENSIONS OF
IMAGING CYTOMETRY ANALYSIS
Ed Luther; Mel Henriksen
55
343/P197. SEMI-MANUAL AND AUTOMATED CLASSIFICATION
OF CIRCULATING CANCER CELLS IN PERIPHERAL BLOOD
VIA HIGH-THROUGHPUT MICROSCOPY
Ramses Agustin; Behrad Azimi; Wenting Shih; Jeffrey
Price
355/P209. FURTHER IMPROVEMENTS IN LED BASED
FLUORESCENCE MICROSCOPY ALLOWED BY
MULTICOLOUR EXCITATION.
Giuliano Mazzini; Marco Angelini; Thomas Beller; Angelo
Paolo Dei Tos; Maria Chiara Giangarè
344/P198. A SIMPLE FLUORESCENCE IMAGING SYSTEM TO
ENUMERATE RESIDUAL LEUKOCYTES IN LEUKODEPLETED
BLOOD
Dania Darleen Yaskanin; James Michael Kelly; JanF Keij;
Mark Carle Connelly; Leon W.M.M. Terstappen
356/P210. A STATISTICAL PATTERN RECOGNITION APPROACH
FOR DETERMINING CELLULAR VIABILITY AND LINEAGE
PHENOTYPE IN CULTURED CELLS AND MURINE BONE
MARROW
John Quinn; Paul W. Fisher; Renold J. Capocasale; Ram
Achuthanandam; Moshe Kam; Peter Bugelski; Leonid
Hrebien
345/P199. ASSESSMENT OF CELL VIABILITY WITH A SIMPLE
IMAGING DEVICE
Steven Gross; Frank Coumans; John Silvia; Mark Connelly;
Leon.W.W.M.Terstappen
346/P200. HIGH-THROUGHPUT CONTINUOUS-MOTION
SCANNING IMAGE CYTOMETER
LamK. Nguyen; Jeffrey Price
347/P201. SIMPLE FLUORESCENCE BASED IMAGE
CYTOMETER
Frank Coumans; Steven Gross; John Verrant; John Sylvia;
Aurel Ymeti; Jan Greve; Leon W.M.M. Terstappen
357/P211. MICROSCALING FLUORESCENT LIFETIME
MEASUREMENTS TO BIOCHIP DEVICES
Rachel J. Errington; Kerenza Njoh; Daniel Matthews; Huw
D. Summers; Paul J. Smith
358/P212. IMAGE DATA EXPLORATION AND ANALYSIS
SOFTWARE
David Basiji; William Ortyn; Cathleen Zimmerman; Richard
Bauer; David Perry; Richard Esposito; Thaddeus George;
Brian Hall; Keith Frost; Philip Morrissey
348/P202. QUANTIFICATION OF ENHANCED GREEN
FLUORESCENT PROTEIN (EGFP) BY LASER SCANNING
CYTOMETRY
Anja Mittag; Birgit Mosch; Thomas Arendt; Attila TáRnok
359/P213. PIXELWISE COMPARISON OF FAST FOURIER
TRANSFORMED IMAGES AS AN EXPLORATORY TOOL FOR
DIFFERENTIAL DIAGNOSIS
Konradin Metze; Randall Luis Adam; Maria Letícia Cintra;
Mariam P. Auada; Rosana C. Morandin; Neucimar Leite
349/P203. MICROVOLUME LASER SCANNING CYTOMETRY
ASSAYS FOR ANALYSIS ONE DAY AFTER SAMPLE
COLLECTION
Vellalore Kakkanaiah; Jun Deng; Aaron B. Kantor
360/P214. ESTIMATION OF IMAGING PRECISION FOR
MICROSCOPE CALIBRATION AND IMAGE COMPRESSION
Bartlomiej Rajwa; Elikplimi Kwaku Asem; J. Paul Robinson;
Tytus Bernas
350/P204. AN EVALUATION OF LED POWERED LIGHT
MICROSCOPE CONVERTED TO DELIVER
EPIFLUORESCENCE TO ENUMERATE CD4 T-CELLS WITH
DYNAL T4 QUANT KIT
Francis Mandy; Peter Stuart Pennefather; Michèle
Bergeron; Chabot Christian; Joanisse Isabelle; Angelini
Marco
361/P215. STUDYING CELL-CELL ADHESION IN HIGHTHROUGHPUT MODE
Natalie Prigozhina; Scott Callaway; Ivana Mikic; Edward
Hunter; Jeffrey Price; Patrick McDonough
351/P205. CONSTRUCTION OF A SIMPLE CONFOCAL
MICROSCOPE WITH EMBEDDED QUANTITATIVE
FLUORESCENCE ANALYSIS
Peng Xi; Silas J. Leavesley; Bartlomiej Rajwa; Tytus Bernas;
Wei-Lin Tiger Bee; Jim Jones; J. Paul Robinson
363/P217. COMPACT MICROSCOPE ON A CHIP
Xin Heng; Xiquan Cui; Demetri Psaltis; Changhuei Yang
352/P206. CYCLINS QUALITATIVE, QUANTITATIVE AND
LOCATION ANALYSIS BY POST-SORTING CONFOCAL
Chun Gao; Jianhong Wu; Deding Tao; Junbo Hu; Yongdong
Feng; Jianping Gong
353/P207. CHARACTERISTICS OF DLP BASED HIGH
THROUGHPUT CONFOCAL MICROSCOPES
Rong Yang; Jeffrey Price; Kristiina Vuori
354/P208. WHOLE-MOUNT IMMUNOFLOURESCENCE
STAINING: AN EN-FACE IDENTIFICATION OF CELL
PROLIFERATION IN RAT HEART ATRIOVENTRICULAR
VALVES
Martin D. Slade; Fang Hengsheng; Jennifer M. Canale;
Timothy P. Reilly; Helen G. Haggerty; Mark G. Mense; Vito
G. Sasseville; Stephen K. Durham; Wendy J. Freebern
56
362/P216. SENSITIVITY OF THE MEAN INTENSITY TO
THRESHOLD
MichaelA. Model; Vera Udovina; James Blank
364/P218. SPECTRAL IMAGING MULTIPHOTON MICROSCOPY
ANALYSIS OF CD36 EXPRESSION WITH QUANTUM DOTS
605 OF 7-KETOCHOLESTEROL-TREATED HUMAN
MONOCYTIC CELLS AND HUMAN ATHEROSCLEROTIC
TISSUE SECTIONS
Edmond Kahn; Vejux Anne; Dumas Dominique; Frouin
Frédérique; Lizard Gerard
365/P219. CYTOSPEC – A PACKAGE FOR MULTISPECTRAL
ANALYSIS OF FLOW AND IMAGE DATA
Valery P. Patsekin; J. Paul Robinson
366/P220. SIMULTANEOUS IMAGING AND CLASSIFICATION
OF ALL CHROMOSOMES IN THE 3D INTERPHASE NUCLEUS
USING SPECTRAL KARYOTYPING
Bart J. Vermolen; Ian Theodore Young; Sabine Mai; Zelda
Lichtensztejn; Yuval Garini
367/P221. INCREASING LEGIBILITY IN HIGHLY
AUTOFLUORESCENT TISSUE FISH SAMPLES
James R. Mansfield; Kenneth Bloom; Richard Levenson
368/P222. QUANTITATIVE HYPERSPECTRAL UNMIXING FOR
REMOVAL OF AUTOFLUORESCENCE FROM WHOLE TISSUE
SECTIONS
Paul Constantinou; David Hedley; Brian Wilson
369/P223. SPECTRAL IMAGING MICROSCOPY WEB SITES AND
DATA
George McNamara; Amit Gupta; James Reynaert; Thomas
D. Coates; Carl A. Boswell
370/P224. DISSECTION OF THE MOLECULAR MECHANISMS
FOR MATRIX PROTEIN TARGETING TO THE PLANT GOLGI
APPARATUS
Loren A. Matheson; Giovanni Stefano; Federica Brandizzi
371/P225. IMAGING TUMORS WITH THREE DIFFERENT
MODALITIES: MAGNETIC RESONANCE IMAGING,
CONFOCAL MICROSCOPY, AND H&E STAINING
Rebecca Milman Marsh; James Andrew Bankson; Nalini
Patel; John Hazle; Nicholas H. A. Terry
372/P226. USING LIVE CELL FLUORESCENT MICROSCOPY TO
MEASURE THE MODULATION OF MICROTUBULE
DYNAMICS BY THE MICROTUBULE-ASSOCIATED PROTEIN
MAP1A IN VIVO
Elliott M. Faller; David L. Brown
373/P227. LIGHT-SCATTERING-BASED MORPHOMETRIC
CELLULAR RESPONSE TO TRANSFECTION BY THE BCL-XL
TRANSMEMBRANE DOMAIN
Nada N. Boustany; Jing-Yi Zheng
374/P241. LUNG EOSINOPHIL OCCURRENCE IN F4/80+ CELLS
Sun-Sang Joseph Sung; Joanne Lannigan
375/P242. MEASURING INDIVIDUAL CELL ACTIVATION AND
INVASION MARKERS, INDICATING POSSIBLE LYMPH NODE
EXPOSURE TO INVASIVE BREAST CANCER CELLS
Naomi Zurgil; Elena Afrimzon; Yana Shafran; Assaf
Deutsch; Pnina Leibovitz; Judith Sandbank; Mordechai
Deutsch
376/P243. CONTROL OF VIREMIA AND PRESERVATION OF
INTESTINAL CD4+ T CELLS IN SHIVSF162P3 INFECTED
MACAQUES INTRAVENOUSLY CHALLENGED WITH
PATHOGENIC SIVMAC251
Bapi Pahar; Mike Piatak; Jeff Lifson; Andrew A. Lackner;
Binhua Ling; Preston Marx; Xiaolei Wang; Ronald S. Veazey
377/P244. ASSESSMENT OF PROTEIN CO-LOCALIZATION
WITH AN IMAGING FLOW CYTOMETER AND NOVEL
ANALYTICAL METHOD
Thaddeus George; Brian Hall; Ronald Taylor; Paul Beum;
Cathleen Zimmerman; David Basiji; Philip Morrissey; Keith
Frost; William Ortyn; Richard Bauer; David Perry; Richard
Esposito
378/P245. MORPHOMETRIC LYMPHOCITE MODEL:
APPLICATION TO THE SCANNING FLOW CYTOMETRY
Loiko Valery; Ruban Gennady; Olga Gritsai; Kosmacheva
Svetlana
379/P246. T CELL DETERMINATION USING MICROCAPILLARY
CYTOMETRY
Kamala Tyagarajan; Dianne Fishwild; Alemayehu Mergia;
Leonard Buchner; Jeff Harvey
380/P247. USE OF FLOW CYTOMETRY ANALYSIS ALONG WITH
AGGREGATION AND MOLECULAR BIOLOGY TO
DETERMINE THE EFFECT OF CLOPIDOGREL IN
NEUROLOGICAL PATIENTS
Agathe Debliquis; Bernard Chatelain; Christian Chatelain;
Patrice Laloux
381/P248. IMMUNOPHENOTIPICAL ALTERATIONS IN T
LYMPHOCYTES FROM PATIENTS WITH CHRONIC RENAL
FAILURE
Angela Hernández; Miguel A. Sánchez; Martin Villarroel;
Manuel Leonardo Acuña; Hugo Barcenilla; David Diaz;
Trinidad Parra; Alfredo Prieto; Miriam Calvino; Jaime
Perez; Jorge Monserrat; Gabriel De Arriba; Eduardo Reyes;
Melchor Alvarez De Mom
382/P249. IMMUNOLOGICAL CHARACTERISTIC OF HUMAN
MAST CELL LINE
Ng Sinnie
383/P250. DISTURBANCES OF COSTIMULATORY NETWORK
ON PERIPHERAL BLOOD T CELLS IN SYSTEMIC LUPUS
ERYTHEMATOSUS AND RHEUMATOID ARTHRITIS
Lanlan Wang; Bei Cai; Jie Chen; Weihua Feng; Lixin Li;
Jiangtao Tang; Honggang Wei; Lijuan Wu
384/P251. GENERATION OF CLONALLY DIVERSE VIRUSSPECIFIC CD8+ T CELL MEMORY OCCURS IN THE CD62LHI
SUBSET EARLY DURING THE EFFECTOR PHASE OF
INFECTION
Kenneth Field; Katherine Kedzierska; Vanessa Venturi;
Misty Jenkins; John Stambas; Miles Davenport; Stephen
Turner; Peter Doherty
385/P252. HIV-1 TAT PROTEIN INDUCES A DECREASE IN BOTH
SURFACE AND INTRACELLULAR CD127 PROTEIN
EXPRESSION IN CD8 T-CELLS WITH A CONCOMITANT
DECLINE IN CD127MRNA TRANSCRIPTS: IMPLICATIONS
FOR CD8 T-CELL FUNCTION IN HIV INFECTION
ElliottM. Faller; Juzer Kakal; Paul Macpherson
386/P253. ROLE OF PROBIOTICS ON STIMULATION OF LOCAL
MAMMARY IMMUNE RESPONSE IN COWS
Mercedes Alonso-Gomez; Fiona Crispie; Katja
Klostermann; William Meany; Sean Arkins
387/P254. CD27, A MARKER TO DISTINGUISH IMMATURE TLINEAGE CELLS BEFORE AND AFTER THE -SELECTION
CHECKPOINT
Rochelle Anne Diamond; Tom Taghon; Mary Yui; Adams
Lura Stephanie; Ellen V. Rothenberg
388/P255. COMPARISON OF T CELL RESPONSES FROM TWO
VACCINE PROTOCOLS USING MULTIFUNCTIONAL
PROFILING
Laurie Lamoreaux; Stephen Perfetto; Mario Roederer;
Richard Koup
389/P256. A POPULATION OF IGD+CD27- B CELLS WITH
SIMILARITIES TO ACTIVATED SPLENIC MARGINAL ZONE B
CELLS IS FOUND IN THE BLOOD OF PATIENTS WITH ACTIVE
SYSTEMIC LUPUS ERYTHEMATOSUS, CORRELATES WITH
DISEASE ACTIVITY, AND IS SENSITIVE TO IL6 RECEPTOR
BLOCKADE
Randy Thomas Fischer; Sarah Okada; Patricia Lugar;
Rebecca Slota; Cheryl Yarboro; Nancy S. Longo; John
Hardin; Gabor G. Illei; Peter E. Lipsky; Amrie C. Grammer
57
390/P257. DIFFERENTIAL EXPRESSION AND ACTIVATION OF
P38-MK2 PATHWAY IN MOUSE BLOOD MONOCYTE
SUBPOPULATIONS
Jingyong Zhao; Lisa Green; Philip Marder; Songqing Na
391/P258. FURTHER PURIFICATION OF HEMATOPOIETIC STEM
CELLS
Yohei Morita; Hideo Ema; Hiromitsu Nakauchi
392/P259. CLUSTERED CARBOHYDRATES AS A TARGET FOR
NATURAL KILLER CELLS: A MODEL SYSTEM
ElenaI. Kovalenko; Elena V. Abakushina; Veena Kapoor;
Elena Y.U. Korchagina; Sergei V. Khaidukov; Irina M.
Molotkovskaya; Alexander M. Sapozhnikov; Pavel A.
Vlaskin; Nicolai V. Bovin; William G. Telford
393/P260. MEASUREMENT OF TWO-DIMENSIONAL LATTICES
USING FLUORESCENCE RESONANCE ENERGY TRANSFER
APPLIED IN FLOW CYTOMETRY
Rob Woestenenk; Arga Staassen; Nancy Jacobs; Jan
Boezeman; Waander L. Van Heerde
394/P261. CHEMOKINE RECEPTOR EXPRESSION DIFFERENCES:
A GROSS PHENOTYPIC ANALYSIS OF THREE MOUSE
STRAINS
James David; Joey Schmuhl; Frank Mortari
395/P262. DEVELOPMENT AND VALIDATION OF AN
INTRACELLULAR CYTOKINE STAINING ASSAY FOR
VACCINE EVALUATION
Stephen C. De Rosa; Yunda Huang; Ya-Lin Chiu; M. Juliana
McElrath; Helen Horton
396/P263. CHARACTERIZATION OF SOLUBLE MEMBRANES
EXPRESSING CD154 TO BE USED THROUGH CD40
BINDING IN THE IN VITRO MODULATION OF HUMAN B
LYMPHOCYTE PROLIFERATION AND DIFFERENTIATION
ÉRic Ducas; Nathalie Dussault; Mathieu Drouin; Daniel
Jung; Sonia Néron
397/P264. PHOSPHATIDYLINOSITOL-3-KINASE-DEPENDENT
ACTIVATION OF ERK BY STEM CELL FACTOR IN PRIMITIVE
BONE MARROW CD34+ CELLS
Frances Tong; Paola Aravena; David Hedley; Robert
Sutherland
398/P265. ANALYSIS OF DAP 12 TRANSMEMBRANE
SIGNALING ADAPTOR PROTEIN EXPRESSION ON
LEUKOCYTE SUBSETS WITH A FIVE COLOR FLOW
CYTOMETRY STRATEGY
Laurent Chiche; Gaëlle Bouvier; Julie Vernier; Frédéric
Vely; Eric Vivier; Félix Montero-Julian
399/P266. DEFINITION OF A MULTIPARAMETER PHENOTYPE
FACILITATING ENRICHMENT OF LIVE HUMAN CD4+
REGULATORY T CELLS
Dennis Hartigan-O’Connor; Ck Poon; Brinda Emu; Jeff
Mold; Elizabeth Sinclair; Jeff Critchfield; Steven G. Deeks;
Joseph M. McCune
400/P267. PLASMA CYTOKINE PROFILE IN HEALTHY INFANTS
IS NOT INFLUENCED BY VACCINATION
Jean-Luc Rummens; Marc Raes; Hanne Jongen; Veerle
Peeters; Petra Scholtens; Brigitte Maes; Karen Hensen
58
401/P268. MULTICOLOR FLOWCYTOMETRIC ANALYSIS OF
HIV-1 INFECTION OF NOD/SCID2MICROGLOBULIN
DEFICIENT MICE XENOGRAFTED WITH HUMAN CORD
BLOOD MONONUCLEAR CELLS
Seiji Okada; Yumi Goto; Hideki Harada; Shinya Suzu
402/P269. TWELVE-COLOR PHENOTYPIC AND FUNCTIONAL
CHARACTERIZATION OF T LYMPHOCYTES AFTER
INFLUENZA VACCINATION
Sandra Yoder; Kathryn M. Edwards; Thomas Talbot; James E.
Crowe; Michael Rock
403/P270. AMINE REACTIVE DYES: AN EFFECTIVE TOOL TO
DISCRIMINATE LIVE AND DEAD CELLS IN
POLYCHROMATIC FLOW CYTOMETRY
Stephen Perfetto; Pratip K. Chattopadhyay; Laurie
Lamoreaux; Richard Nguyen; David Ambrozak; Koup A.
Richard; Mario Roederer
404/P271. MICE AND MEN I: NAIVE AND MEMORY NATURAL
KILLER (NK) AND OTHER CELLS OF THE INNATE IMMUNE
SYSTEM
Doug Redelman
405/P272. MICE AND MEN II: LABORATORY MICE LACK
MEMORY CYTOTOXIC T CELLS
Doug Redelman; Kory Alderson; Dorothy Hudig; William J.
Murphy; David Tamang
406/P273. MICE AND MEN III: LABORATORY MICE LACK
CYTOLYTICALLY ACTIVE NATURAL KILLER (NK) CELLS
COMPARABLE TO THOSE FOUND IN HUMANS
Doug Redelman; Kory Alderson; Will Hallett; Dorothy
Hudig; William J. Murphy; David Tamang
407/P274. MICE AND MEN IV: MYELOID CELLS FROM
NORMAL HUMAN SUBJECTS AND FROM LABORATORY
MICE DIFFER IN NUMBER AND IN PHENOTYPE
Doug Redelman; Ken Hunter; Vanessa Kim Berner
408/P275. SUMMARY OF THE ANIMAL HOMOLOGUE SECTION
OF HLDA8
Armin Saalmüller; JoanK. Lunney; Claudia Daubenberger;
William C. Davis; Uwe Fischer; Thomas W. Goebel; Philip
Griebel; Enoc Hollemweguer; Todd M. Lasco; Richard K.
Meister; Hans-Joachim Schubert; Karol Sestak; Paul Sopp;
Falko Steinbach; Wu Xiao-Wei; Bent Aasted
409/P276. TRACING THE DYNAMICS OF THE CELLULAR
PLAYERS OF THE GERMINAL CENTER REACTION
Nicole Wittenbrink; Anke Klein; Johannes Schuchhardt;
Michal Or-Guil
410/P277. EFFECT OF CD4+ T CELL COMPETITION ON
CLONAL EXPANSION AT PHYSIOLOGICAL PRECURSOR
FREQUENCIES
AdrianL. Smith; Barbara Fazekas De St. Groth
411/P278. PROGRESS IN THE DISCOVERY AND DEFINITION OF
MONOCLONAL ANTIBODIES FOR USE IN FELINE
RESEARCH
Richard K. Meister; Karin Taglinger; Karin Haverson;
Nicholas Strohminger; Lawrence E. Mathes
412/P279. INTEGRATED ANALYSIS OF ELECTRONIC VOLUME,
SIDE SCATTER AND PHENOTYPE MARKER EXPRESSION:
APPLICATIONS IN RESEARCH FLOW CYTOMETRY.
Raquel Cabana; Maria Daly; Mark Cheetham; Vincent
Shankey; Richard Thomas; Awtar Krishan
413/P280. AUTOMATION: AN APPROACH TO THE
STANDARDIZATION OF FUNCTIONAL IMMUNE CELLBASED ASSAYS
Julie G. Wilkinson; Cecilia Smith; SybilS. D’Costa; Enrique
Rabellino
425/P301. FK506 REGULATORY EFFECT ON THE EXPRESSION
OF CD28 ACD152 AND ICOS ON T LYMPHOCYTES IN
ALLO-LIVER RECIPIENTS
Lanlan Wang; Yangjuan Bai; Maozhi Liang; Bei Cai; Jie
Chen; Weihua Feng; Jiangtao Tang; Lixin Li; Yongkang Wu
414/P281. CYTOMYC STUDY OF THE EFFECT OF THE PROCESS
OF HEMODIALYSIS ON THE LYMPHOCYTE SUBSETS IN
PATIENTS WITH CHRONIC RENAL FAILURE
Angela Hernández; Miguel A. Sánchez; Martin Villarroel;
Manuel Leonardo Acuña; Hugo Barcenilla; David Diaz;
Trinidad Parra; Jaime Perez; Miriam Calvino; Gabriel De
Arriba; Alfredo Prieto; Jorge Monserrat; Eduardo Reyes;
Melchor ÁLvarez De Mon
426/P302. COMPARING TWO FLOW CYTOMITRY ASSAYS
WITH MTT ASSAY AS SUSCEPTIBILITY TESTS ON ANTITUMOR DRUGS
Jing Yao; Xiaolan Li; Hui Xiao; Peng Zhang; Jianping Gong
415/P282. ROLE OF INTERLEUKIN-3 RECEPTOR IN MYELOID
CELL DIFFERENTIATION
Douglas A. Weidner; James A. McCubrey
416/P283. PHENOTYPIC AND FUNCTIONAL EVALUATION OF
CORD BLOOD T LYMPHOCYTES
Thomas Mc Closkey; Olga Aroniadis; Raj Pahwa; Savita
Pahwa
417/P284. ASSESSING THE REAL SIZE DISTRIBUTION OF
GERMINAL CENTERS
Nicole Wittenbrink; Anke Klein; Johannes Schuchhardt;
Michal Or-Guil
418/P285. HI-DIMENSIONAL FACS ANALYSES OF
DEVELOPMENTAL AND FUNCTIONAL B CELL SUBSETS
James W. Tung; David Parks; Wayne A. Moore; Leonard
Herzenberg; Leonore A. Herzenberg
419/P286. MEASURING THE PHYSIOLOGICAL EFFECTS OF
RNAI IN A HETEROGENEOUS POPULATION OF NONADHERING CELLS AT THE SINGLE CELL RESOLUTION USING
THE OPTICAL LIVECELL ARRAY TECHNOLOGY
Israel Biran; Sarah Haigh; Naomi Zurgil; Motti Deutsch;
Orian Shirihai
420/P287. BLOOD LEUKOCYTE PRODUCTION OF REACTIVE
OXYGEN SPECIES IN PEOPLE WITH TYPE 2 DIABETES
Judith C. Stewart; Donna Speers; Mark W. Frampton
421/P288. THE T LYMPHOCYTE SUBSET AND ACTIVATED T
CELL AFTER TREATMENT OF MMF COMBINATION WITH
CSA IN PATIENTS OF GRAFT-VERSUS-HOST DISEASE
Xin Du; Jianjun Zhang; Jianyu Weng
422/P289. FETAL CELL COUNT™ - ROUTINE DETECTION AND
QUANTIFICATION OF FETAL RED BLOOD CELLS IN
MATERNAL BLOOD BY FLOW CYTOMETRY
J.H.N. Schuitemaker
423/P290. ENUMERATION OF NRBC USING A NOVEL NO-LYSE,
SINGLE PLATFORM FLOW CYTOMETRIC ASSAY
Christine Hopkins; Joanne Luider; Iwona Auer
424/P291. POLYCHROMATIC (8-9 COLOR) FLOW CYTOMETRY
ANALYSIS OF TUMOR ANTIGEN SPECIFIC CD8+ MEMORY T
CELLS: APPLICATION OF QDOT IMMUNOFLUORESCENCE
WITH INTEGRATED DATA REDUCTION USING HYPERLOG™/
FCOM™ AND CLUSTER ANALYSIS
Dan Haley; William Miller; Ulf Petrausch; Kevin Floyd;
Edwin Walker
427/P303. USEFULNESS OF THE ORAL EPITHELIA CELLS’
APOPTOSIS RATE IN NUTRITIONAL ASSESSMENT
Xuelai Luo; Yi Zhou; Yongdong Feng; Deding Tao; Peng
Zhang; Jianping Gong
428/P304. FEASIBILITY OF JAK2 V617F MUTATION DETECTION
IN THE DIFFERENTIAL DIAGNOSIS OF POLYCYTHAEMIA
Jean-Luc Rummens; Karen Hensen; Ariane Luyckx; Rita
Smets; Sabine Franke; Greet Bries; Veerle Peeters;
Reinoud Cartuyvels; Brigitte Maes
429/P305. DEVELOPMENT OF STANDARDIZED FLOW
CYTOMETRY TOOLS TO ASSESS IMMUNOPHENOTYPING IN
IMMUNOTOXICOLOGY STUDIES
Laetitia Roqueblave; Thierry Ramananarivo; Christophe La
Boulaire; Michel Herbert; Félix A. Montero-Julian; Christine
Fornelli
430/P306. FLOW CYTOMETRIC QUANTITATION OF
APOPTOSIS OF CIRCULATING LYMPHOCYTES DURING
PEDIATRIC CARDIAC SURGERY
Jozsef Bocsi; Joerg Hambsch; Peter Schneider; Attila
TáRnok
431/P307. THE USE OF FLOW CYTOMETRY TO MEASURE
INDIVIDUAL RADIOSENSITIVITY AND PREDICT NORMAL
TISSUE LATE TOXICITY FROM RADIOTHERAPY
Kara L. Schnarr; Ian Dayes; Nicole McFarlane; Douglas R.
Boreham
432/P308. FLOW CYTOMETRIC ANALYSIS OF MONOCYTE
INTRACELLULAR PHOSPHO-MAPKAPK2—A ROBUST
MULTI-SPECIED P38 BIOMARKER
Lisa Green; Rita Bowers; Jingyong Zhao; Kelli Brune; Todd
Christopher; Songqing Na; Jeff Wolos; Robert Campbell;
Philip Marder
433/P309. DEVELOPMENT OF A FLOW CYTOMETRIC ASSAY TO
EVALUATE NAIVE AND MEMORY T CELL POPULATIONS IN
HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
Lisa Patti-Diaz; DiannaY Wu
434/P310. ABNORMAL ICAM-1 (CD54) EXPRESSION ON
CIRCULATING PERIPHERAL BLOOD LYMPHOCYTES IN
PATIENTS WITH JUVENILE DERMATOMYOSITIS
Maurice R.G. O’Gorman; KevinA. O’Connor; Lauren M.
Pachman
435/P311. ß-GAL DETECTION UTILIZING A RED
CHROMOGENIC ß-GALACTOSIDE SUBSTRATE WITH FLOW
CYTOMETRY
Lora Wiltshire Barsky; C. Bart Rountree; Kasper Wang; Gay
M. Crooks
59
436/P312. FLOW CYTOMETRIC ANALYSIS OF HUMAN
SPERMATOZOA: THE RELEVANCE FOR INFERTILITY
TREATMENT
Miloslav Suchanek; Marie Havranova; Petra Mrkvickova;
Olina Tepla; Jana Peknicova
447/P323. AN ESSENTIAL ROLE FOR PTEN IN HEMATOPOIETIC
STEM CELL MAINTENANCE, LINEAGE CHOICE, AND
LEUKAEMIA PREVENTION
Jiwang Zhang; Xi He; Jason Ross; Sach Jayasinghe; Jeff
Haug; Leanne Wiedemann; Hong Wu; Linheng Li
437/P313. FLOW CYTOMETRIC AND IMMUNOHISTOLOGICAL
STUDIES ON PROLIFERATING ACTIVITIES IN RECURRENT
MENINGIOMA
Keiji Kawamoto; Hideyuki Oshige; Harubumi Kasai; Takashi
Ryu; Keiichi Azuma; Qiang Li; Takahiro Yamahara; Yoshihiro
Numa
448/P324. TRANSPLANTATION OF EX VIVO EXPANDED LIN–
CELLS SELECTED FROM AUTOLOGOUS PBSC GRAFTS IN
PATIENTS WITH DIAGNOSIS OF LYMPHOMA
Martin Klabusay; Zdenek Koristek; Viera Kohutova;
Jaroslava Vinklarkova; Jiri Mayer
438/P314. CELLULAR PHENOTYPING OF MULTIPLE SCLEROSIS:
PHARMACODYNAMIC CHANGES WITH IFN-BETA-1A
(AVONEX®) TREATMENT
Jun Deng; Vellalore Kakkanaiah; SusanE. Goelz; Donald
Bennett; Aaron B. Kantor
439/P315. BIOMARKERS IN THE DETECTION OF
PRECANCEROUS/CANCEROUS CERVICAL CELLS
Wei-Lin Tiger Bee; Dominik Lenz; Jian Ling; J. Paul
Robinson
440/P316. A LIBS METHOD FOR FLOW CYTOMETRIC
DETERMINATION OF A4B1 RECEPTOR OCCUPANCY AS A
PHARMACODYNAMIC MARKER IN CLINICAL TRIALS
Christopher Vandevert; Ted Yednock
441/P317. ELAVL4: A NEW MOLECULAR MARKER FOR
DETECTION OF DISSEMINATED NEUROBLASTOMA CELLS
USING REAL-TIME QUANTITATIVE RT-PCR
Katrien Swerts; Barbara De Moerloose; Catharina Dhooge;
Yves Benoit; Geneviève Laureys; Jan Philippé
442/P318. FACTOR XIIIA COULD PLAY A ROLE IN THE
VASCULARIZATION OF DISEASED HUMAN CORNEAS
Lili Takács; Gergely Losonczy; Elza Friedländer; Ágnes
Orosz; Enikö Tóth; László Muszbek; András Berta; György
Vereb
443/P319. ELEVATED LEVELS OF CIRCULATING
MICROPARTICLES AND THEIR RELATIONSHIP TO
HYPERCOAGULABLE STATE IN THALASSEMIA PATIENTS
Kovit Pattanapanyasat; Siriphan Gonwong; Egarit Noulsri;
Porntip Chaichompoo; Surada Lerdwana; Suthat
Fucharoen
444/P320. PROTEOMIC ANALYSIS AND REGENERATIVE
POTENTIAL OF THE SIDE POPULATION STEM CELLS FROM
MURINE BONE MARROW AND HUMAN FETAL LIVER
Gopal Pande; Azra Fatima; Saritha Cv; Charumathi A;
Srinivas G
445/P321. EARLY ENGRAFTMENT OF CIRCULATING
DENDRITIC CELLS AFTER HEMATOPOIETIC CELL
TRANSPLANTATION IN PATIENTS TREATED BY REDUCEDINTENSITY CONDITIONING REGIMENS (RICR)
Francis Belloc; Reza Tabrizi; Virginie Perreau; Xavier
Lafarge; François Comeau; Francis Lacombe; Jean-Michel
Boiron
446/P322. HEMATOPOIETIC CELL TRANSPLANTATION IN
HUMANS RESULTS IN GENERATION OF DONOR-DERIVED
EPITHELIAL CELLS, HOWEVER THEIR IDENTIFICATION
REQUIRES STRINGENT DETECTION SYSTEMS
Marie Follo; Vanessa Deggim; Yannis Metaxas; Philipp
Faber; Alexandros Spyridonidis
60
449/P325. IT’S NOT JUST FOR MICE: A PRACTICAL GUIDE TO
IDENTIFYING AND SORTING “SP” CELLS FROM VARIOUS
SOURCES
William J. Murphy; Andrew D. Bantly; Charles H. Pletcher;
Jonni S. Moore
450/P326. FLAER AS A SENSITIVE MARKER IN THE FLOW
CYTOMETRIC EVALUATION OF PNH
Veronique Stove; Luc Noens; Jan Philippé
451/P327. COMPARATIVE EVALUATION OF THREE FLOW
CYTOMETRIC TECHNIQUES FOR CD34 ENUMERATION IN
VARIOUS SAMPLE TYPES
János Kappelmayer; Valéria Sziráki Kiss; Éva Karászi
452/P328. A COST EFFECTIVE TECHNOLOGY FOR ABSOLUTE
CD4 T-CELL ENUMERATION USING THE HYBRID FLOW
CYTOMETRIC PLATFORM
Tao Ding; MichèLe Bergeron; George Janossy; Rudi Varro;
Francis Mandy
453/P329. FLOW CYTOMETRY ASSESSMENT OF THE EFFECTS
OF NALOXONE ON HUMAN PHAGOCYTIC CELLS
Bernard Delvaux; Bernard Chatelain; Jacques Jamart;
Nicolas Neyman; Etienne Installé; Marc De Kock
454/P330. NOVEL FLOW CYTOMETRIC ASSAY USING KI-67
AND PCNA FOR THE DETERMINATION OF T- AND B-CELL
LYMPHOCYTE PROLIFERATION AFTER MITOGEN
STIMULATION
Patricia Johnson; Joanne Luider; T Bowen; Iwona Auer
455/P331. COMPARISON OF HUMAN IMMUNODEFICIENCY
VIRUS-1-SPECIFIC CD4+ AND CD8+ T CELL RESPONSES IN
ADULTS IN SUB-SAHARAN AFRICA
Sharon Shalekoff; Stephen Meddows-Taylor; Glenda Gray;
Louise Kuhn; Gayle Sherman; Ashraf Coovadiaah; Caroline
Tiemessen
456/P332. AGE DEPENDENCY AND MUTUAL RELATIONS IN TAND B- LYMPHOCYTE ABNORMALITIES IN CVID PATIENTS
Marcela Vlkova
457/P333. APPLICATION OF BASOPHIL ACTIVATION TEST ON
ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS
DIAGNOSIS AND MONITORING
Marianna Tzanoudaki; Anna Katellari; Manolis Liatsis;
Stavros Doudounakis; Effie Vrachnou; Maria Kanariou
458/P334. MULTI-CENTER ANALYTICAL PERFORMANCE OF
CMV SPECIFIC CD8+ T LYMPHOCYTES USING A
STANDARDIZED SINGLE-PLATFORM HLA TETRAMER ASSAY
Michael Keeney; Jan Popma; Karin Weir; Kristen White;
Jeremy Smith; Kurtis Bray; Lori Lofaro; Paula Southwick;
MichaelJ. Boeckh; Ian Chin-Yee; Christopher Boyce
459/P335. CYTOMETRIC ANALYSIS OF A NOVEL,
AUTOLOGOUS CELL CULTURE SYSTEM CAPABLE OF
PRODUCING HIGH-LEVELS OF AUTOLOGOUS HIV-1 FOR
INDIVIDUALIZED THERAPEUTIC VACCINATION
Charles Mac Trubey; Jeffrey D. Lifson
460/P336. HIGH-SPEED QUANTISATION OF CMV SPECIFIC
CYTOTOXIC T-CELLS IN WHOLE BLOOD
Lene Have Poulsen; Kivin Jacobsen; Ian Storie; Jesper
Laursen
461/P337. SUPPRESSOR FUNCTION OF CD4+CD25+
REGULATORY CELLS IN COELIAC DISEASE
Marilena Granzotto; Sara Quaglia; Alberto Tommasini;
Gianni Presani; Tarcisio Not; Stefano Martellossi;
Alessandro Ventura
462/P338. WITHDRAWN
463/P339. MULTIPLEXED MEASUREMENT OF CYTOKINES
SECRETION IN TEARS OF PATIENTS WITH TOPICAL ANTIGLAUCOMA TREATMENTS BY USE OF THE BIO-PLEX
SUSPENSION ARRAY SYSTEM
Laure Malvitte; Thomas Montange; Corinne Joffre; Alain
Bron; Catherine Creuzot-Garcher; Gérard Lizard
464/P340. CHARACTERISATION OF STROMAL CELL SUBSETS
USING CONFOCAL MICROSCOPY AND FLOW CYTOMETRY
Debbie L. Hardie; Tie Zheng Hou; Sian Lax; Darrell Pilling;
Ed Rainger; Hans-Jorg BüHring; Christopher Dominic
Buckley
465/P341. ASSOCIATIONS BETWEEN T/T GENOTYPE OF THE
CD14 GENE´S C(-159)T POLYMORPHISM, SERUM SCD14
CONCENTRATION, SCD14 ISOTYPE DISTRIBUTION AND
DISEASE ACTIVITY AND COURSE IN PATIENTS SUFFERING
FROM POLYMYOSITIS/DERMATOMYOSITIS (PM/DM)
Andrea Sümegi; Katalin Dankó; Andrea Ponyi; Gyula
Szegedi; Péter Antal-Szalmás
466/P342. LEUCOCYTE ALKALINE PHOSPHATASE ANALYSIS
BY FLOW CYTOMETRY IN PATIENTS WITH CHRONIC
MYELOID LEUKEMIA
Tania Tieko Takao; Pedro Enrique Dorlhiac - Llacer; Elvira
Velloso; Marcos Antonio Munhoz; Maria Mirtes Sales
467/P343. REASSESSING CELL-CYCLE SPECIFICITY OF
ANTICANCER DRUGS BY A NOVEL FLOWCYTOMETRY
ASSAY
Peng Zhang; Deding Tao; Daxing Xie; Yongdong Feng;
Jianhong Wu; Junbo Hu; Jianping Gong
468/P344. ZAP-70 IN B-CLL, MEASURED BY FLOW
CYTOMETRY AND RT-PCR, AND AGREEMENT WITH
MUTATIONAL STATUS AND OUTCOME
Jan Philippe; Femke Van Bockstaele; Ann Janssens; Fritz
Offner; Bruno Verhasselt
469/P345. A SINGLE TUBE ANTIBODY PANEL FOR LEUKOCYTE
DIFFERENTIAL IN BLOOD OR BONE MARROW
Sven Bjornsson; Saga Wahlström; Ingela Bernevi; Per
Simonsson
470/P346. MEASUREMENT OF ZAP-70 EXPRESSION IN CLL
USING AN OPTIMIZED FLOW CYTOMETRIC ASSAY FOR
ZAP-70 PROTEIN LEVELS IN WHOLE BLOOD SAMPLES
Vincent Shankey; Paul Scibelli; Jeffery Cobb; Cecilia Smith;
Rhonda Mills; Meryl Forman
471/P347. HAEMOPHAGOCYTIC LYMPHOHISTOCYTOSIS
ASSOCIATED TO A MONOCLONAL EXPANSION OF EBV
NEGATIVE NK CELL
Maria Rocio Lopez Alvarez; Maria Gema Salgado Cecilia;
Ana Galera MiñArro; Carmen Botella Martinez; Maria
Victoria Martinez Sanchez; Jose Luis Fuster Soler; Damia
Heine SuñEr; Manuel Muro Amador; Maria Rocio Alvarez
Lopez; Ana Maria Garcia Alonso; Alfredo Minguela-Puras
472/P348. ZAP-70 EXPRESSION IN HEMATOGONES OR
PRECURSOR B CELLS
Michael Schwartz; Bruce H. Davis
473/P349. RESIDUAL LEUKEMIA CELLS MONITORING BY
MULTICOLOR FLOW CYTOMETRY – A PILOT STUDY OF RQPCR ASSISTED ANTIBODY PANEL DESIGN
Tomas Kalina; Ester Mejstrikova; Sona Hubackova; Eva
Fronkova; Daniel Thürner; Pavel Semerak; Jan Trka; Ondrej
Hrusak
474/P350. IDENTIFICATION AND CHARACTERIZATION OF A
CD20 POSITIVE T-CELL PROLYMPHOCYTIC LEUKAEMIA (TPLL) AS A RESULT OF TRIPLE COLOUR FLOW CYTOMETRIC
ANALYSIS
Oktavia Jonasdottir; Gitte Olesen; Marianne Thisted; Tom
Just
475/P351. DENSITY OF CD52 ANTIGEN ON LYMPHOCYTES,
CD34+ CELLS FROM GRAFT OF PERIPHERAL BLOOD STEM
CELLS AND TUMOR CELLS FROM PATIENTS WITH
CHRONIC B-CELL LYMPHPPROLIFERATIVE DISEASES
Martin Klabusay; Vera Sukova; Petr Coupek
476/P352. OPTIMIZING FLOW CYTOMETRIC DETECTION OF
ZAP-70 EXPRESSION IN CLL CELLS
Sergey N. Preobrazhensky; David W. Bahler
477/P353. CORRELATION OF FLOW CYTOMETRIC ZAP-70
RESULTS USING A MODIFIED FIXATION/
PERMEABILIZATION KIT WITH SOMATIC HYPERMUTATION
DATA
Joanne Luider; Howard Wong; Patricia Johnson; Faith
Jahelka; Marco Perizzolo; Iwona Auer; Adnan Mansoor
478/P354. IMMUNOPHENOTYPIC FEATURES OF ACUTE
MYELOID LEUKEMIA WITH AML1/ETO FUSION GENE
Jianjun Zhang; Xin Du
479/P355. TELOMERE LENGTH AS PREDICTIVE FACTOR IN BCELL CHRONIC LYMPHOCYTIC LEUKAEMIA (B-CLL)
Heike Himmelreich; Alexander Roeth; Jan Duerig; Sybille
N. Matthey; Gabriela M. Baerlocher
480/P356. ABNORMALITIES OF BONE MARROW MONOCYTES
IN MYELODYSPLASTIC SYNDROMES
Irene Lorand-Metze; Elisangela Ribeiro; Carmen S. P. Lima;
Konradin Metze
481/P357. TWO CASES OF CHRONIC LYMPHOCYTIC
LEUKAEMIA (CLL) IN YOUNG ADULTS DIAGNOSED BY
FLOW CYTOMETRY
Maria Cristina F. Marques; Jorge Rolao Candeias;
Conceição Magalhães; Sofia Meirinho; Sónia Fonseca;
Cláudio Reis
61
482/P358. APPLICATION OF A SET OF INTRACELLULAR TRIPLE
COLOUR REAGENTS FOR FLOW CYTOMETRIC ANALYSIS
OF LEUKAEMIA
Oktavia Jonasdottir; Gitte Olesen; Marianne Thisted; Tom
Just
483/P359. ANALYSIS OF SURFACE AND SOLUBLE L-SELECTIN
IN RELATION TO FLOW CYTOMETRIC PROGNOSTIC
MARKERS IN CHRONIC LYMPHOCYTIC LEUKEMIA
Flora Kiss; Ildiko Trefan; Janos Kappelmayer
484/P360. STUDY OF MORPHOLOGIC AND
IMMUNOPHENOTYPIC PROFILE OF LYMPHOCYTES ON
PERIPHERAL BLOOD FROM PATIENTS WITH MYCOSIS
FUNGOIDES IN EARLY AND ADVANCED STAGES
Patrícia Aparecida Ferreira Oliveira; Maria Mirtes Sales;
Leila Antonangelo; Leila Jaldim Borracha Gonçalves;
Tatiana Kovach Hayashida; José Antonio Sanches Jr
485/P361. CYTOFLUOROGRAPHIC IDENTIFICATION OF
HEMATOPOIETIC NEOPLASMS AND INFLAMMATORY
DISORDERS IN HUMAN VITREOUS FLUID
Phillip Ruiz; Senen Rodriguez; Alex Amador; Nubia
Herrera; Janet Davis
486/P362. RETINOIC ACID COMBINATION WITH TARGET
REAGENTS INDUCING CELL APOPTOSIS IN ORAL
SQUAMOUS CELL CARCINOMA
Wan-Tao Chen; Ping Zhang; Qin Xu; Zhiyuan Zhang;
Xiaojian Zhou; Ronggen He
62
487/P363. FLOW CYTOMETRIC MONITORING OF NUCLEAR
MARKER EXPRESSION IN CELLS FROM BODY CAVITY
FLUIDS OF CANCER PATIENTS
Awtar Krishan; Ronald Hamelik; Malancha Sarkar
488/P364. NO OR LOW EXPRESSION OF CYCLINS (A,B1,D1,E)
IN GASTROENTERIC TUMOR
Yi Zhou; Haocheng Long; Weihua Li; Deding Tao; Junbo
Hu; Jianping Gong
489/P365. INDUCE DIFFERENTIATION WITH ALL-TRANS
RETINOIC ACID IN HUMAN NEUROBLASTOMA CELLS
Xi-Hua Wang; Meng-Yuan Yue; Jing-Yan Tang; Wan-Tao
Chen
490/P154A. GLOBAL ANALYSIS OF CELL TYPE-SPECIFIC GENE
EXPRESSION AND REGULATION
David W. Galbraith; Changqing Zhang; Georgina Lambert;
Roger A. Barthelson
491/P167A. DEVELOPMENT OF HIGH-PRODUCER CHO CELLS
BY FLOW CYTOMETRY
Yael Gothelf; Mira Toister-Achituv; Daniel Helman; Moshe
Smolarsky
Sunday, 21 May
Workshops
16:00 - 17:30
1. Cell-Based Imaging Using High Content
Screening and Analysis ............... Room 202
4. Flow Cytometric Analysis of Leukemia and
Lymphoma................................... Room 302
Chair: Joe Trask
Co-chairs: Mike Borowitz and Brent Wood
High Content Screening (HCS) and High Content Analysis (HCA)
also known as automated microscopy image analysis has
rapidly evolved from its infancy and has redefined the way
research is being performed from basic science to
pharmaceutical drug development. HCS/HCA is based on live
or fixed cells adhered to glass slides or microtiter plates that
rapidly measure morphological characteristics, cell cycle,
apoptosis, toxicity, protein redistribution, receptor
internalization, and other cellular processes using sophisticated
image analysis algorithms. It is the goal of this high-content
imaging workshop to present instrumentation used, the ever
important informatics component, other critical aspects of the
technology, and case studies based on submitted abstracts
and speakers. The workshop will provide informative
discussions from basic science research to drug development
that includes siRNA, target identification, target validation,
screening, lead optimization through preclinical studies. A
recent issue in this technology is lack of standards (image
format, calibration, algorithms, etc.) that we hope to briefly
discuss.
This workshop will discuss multiparameter analysis in
immunophenotyping in leukemia and lymphoma. Emphasis
will be placed on interpretation of visual displays of
immunophenotyping data, and on methods to extract the
maximum amount of information from specimens of
hematolymphoid tissue. Description of patterns seen in normal
bone marrow and lymphoid tissues will be illustrated as a basis
for interpreting abnormal specimens. Selected cases will be
chosen to illustrate a range of hematologic neoplasias,
including both straightforward and complex cases. The power
of the technology for the detection of minimal residual disease
will be demonstrated, and clinical relevance of findings
discussed. Participants are encouraged to bring dot plots of
interesting and challenging cases in this area as potential topics
for discussion.
2. Hot Topics in Apoptosis .............. Room 202
Chair: Andrea Cossarizza
The session will use brief presentations form selected abstracts
to focus discussion on some new aspects of apoptosis, paying
particular attention to new technologies used to investigate
this complex phenomenon, as well as on novel pathways and
molecules involved in cell death.
3. Biosafety ...................................... Room 301
Chair: Ingrid Schmid
As flow cytometry continues to grow in importance as an
analytical tool for the analysis of biological samples containing
either known or unknown pathogens safety measures to
protect flow cytometer operators from hazards are of
paramount importance. Workshop focus areas for discussion
will be a review of the results from a survey on current
biosafety practices in Shared Flow Cytometry Resource
laboratories conducted by the ISAC Biosafety Committee in
2005, the draft version of the up-dated ISAC Biosafety
Guidelines for Sorting of Unfixed Cells, information about World
Health Organization and European Community biosafety
resources and references, and future directions for the ISAC
Biosafety Committee.
5.
Flow Technology Instrument
Developments ............................. Room 303
Chair: John Nolan
Recent trends in flow cytometry instrumentation are resulting
in smaller instruments with increased measurement
capabilities. The continued incorporation of small, low power
light sources and detectors together with increased use of
microfabrication have the potential to lead to a new generation
of low cost instruments. At the same time, the optical
measurement capabilities of flow-based platforms are
increasing, making new applications feasible. Continued
increases in serial and parallel measurement throughput raise
issues of data handling and analysis. This workshop will focus
on emerging trends in flow cytometry instrumentation, as well
as the applications that are driving them. Brief presentations
will serve to stimulate discussion.
6. Cytometry Education .................. Room 304
Chair: Lori Krueger
ISAC’s diverse membership base has diverse education and
training needs. ISAC members include individuals working in
Academic and Clinical Research and Biopharma and Biotech
laboratories in both resource rich, and resource poor nations.
ISAC has provided educational opportunities to its members
through participation in its international Congresses, its journal,
and through ISAC-sponsored meetings and seminars. However,
to meet the needs of our growing cytometry community,
additional educational tools are needed. A brief overview of
the ISAC Education Plan will be presented. In addition, this
workshop invites your participation in identifying the
educational needs of cytometrists from all scientific fields from
all parts of the globe.
63
Tuesday, 23 May
Workshops
7. Cytometric Approaches for Biomarker
Research ....................................... Room 202
Chair: Phil Marder
This workshop will provide an overview description of what a
biomarker is and how cytometric technology has impacted
this nascent discipline. Potential areas for discussion will be:
identification of disease-associated biomarkers, use of
biomarkers for selection of patient subgroups, therapeutic
target identification biomarkers in preclinical and clinical
studies, biomarkers for tracking pharmacologic effects before
uncovering therapeutic efficacy, and issues surrounding
standardization and validation of biomarker methodologies. A
group of panel participants will be invited from the submitted
Congress abstracts and also from others who might have
relevant data to share.
8. Image Segmentation .................. Room 301
Chair : Jelena Kovacevic
Automated analysis of cell images has become an important
part of both applied and basic cytometry. For many applications,
accurate identification of single cell regions is critical. This can
be quite difficult and a range of approaches have been used in
the past with varying success. This workshop will focus on
exciting recent work on this important problem.
9. Multiplexed and Microparticle
Assays .......................................... Room 302
Chair: Marie Iannone
Microsphere-based flow cytometric analysis is a sensitive,
efficient and increasingly popular format for analyte
measurement. In addition, bead-based approaches may also
be used to assess other biomolecular measurements including
enzymatic activity, nucleic acid measurement and molecular
interactions. This workshop will focus on factors that impact
the sensitivity and the design of these assays. There will be an
opportunity for the discussion of issues and a chance to ask
questions.
10. Cell Functional Analysis ............... Room 303
Co-chairs: George Babcock and Padma Narayanan
This workshop is intended to allow participants to discuss
functional analysis in the broadest sense. This would include
methodologies to measure cell function (both new and old),
data analysis relating to particular methods, mechanisms
relating to the measurement of cell functions, and novel
approaches to cell functional problems. Included (but not
restricted to) are functional analyses of prokaryotic and
eukaryotic cells, DNA, beads, and other particles. Discussion of
new or modified instrumentation to measure function would
64
16:00 - 17:30
also be encouraged. Although analysis using flow and image
methods will be encouraged, functional analysis using other
methods are also possible discussion topics. Although this
workshop is not intended for presentations stressing research
data, participants may use their data as examples or to make
specific points. Examples of specific problems encountered
related to the topics listed above will also be appropriate for
discussion. Attendees are urged to participate in the
discussions, i.e. join in on the fun.
11. Spectroscopy Issues and
Cytometry .................................... Room 304
Co-chairs: Jeremy Lerner, Steven Locket, and
Robert Zucker
Spectroscopy has been applied to imaging and flow based
systems. Many confocal microscope systems have the potential
to measure spectral properties of dye molecules located in
tissues. Some can very effectively use unmixing algorithms to
separate fluorescence probes that have overlapping spectra.
There is much that can be gained by studying changes in spectra
under different conditions. Methods to calibrate the spectral
systems will be discussed as well as processing this complex
data after it is obtained. This workshop will focus on all issues
involving spectroscopy in flow, confocal and other imaging
systems. Potential applications will also be discussed.
12. Cytometry Applications in
Biotechnology ....................... Room 205 B/C
Chair: Gary Elliot
As pharmaceutical and biotechnology companies work to
develop ever more sophisticated medicines, it becomes
increasingly important to access the latest available
technologies and assay methods for use in screening, validation
and characterization of lead molecules. In order to develop
more targeted therapeutics while limiting off-target effects,
the pharmaceutical and biotech industry is making increased
use of highly multiplexed assay formats, high content
approaches such as imaging cytometry and integration of
multiple technology platforms. This workshop will focus on
the application of new assay methods and new cytometric
technologies to enhance drug discovery and development.
Topics for discussion will be generated from submitted
Congress abstracts and will include novel flow cytometry
methods, new cytometer/imaging platforms and the impact
of nanotechnologies in therapeutic drug development.
Wednesday, 24 May
Workshops
16:00 - 17:30
13. Tracking Microorganisms at the Single Cell
Level: Industrial, Environment, and Aquatic
Biology ......................................... Room 202
15. Topics in Multicolor Immunofluorescence:
Appropriate Use of Isotype
Controls ....................................... Room 302
Chair: Michel Denis
Co-chairs: Ruud Hulspas and Lori Krueger
A few topics (up to 5 or so) will be selected from oral and
poster presentations. The presenting authors will be asked to
define in 5 minutes or less a topic (question, finding, etc.)
submitted to discussion. The debate will be opened for about
15 minutes and then another topic will be addressed. Some
flexibility may be applied to adjust to the interest of the
audience. Among possible topics, the following might be
considered:
• Descriptive aspects: detection and counting of
microorganisms by using flow cytometry in aquatic
environments (seawater, fresh water), in the industry and
other environments;
• Spatial and temporal variability of microorganism
distributions;
• Cell viability;
• Automated in situ observation;
• Microbes as bioindicators and/or biosensors?
• What should be a research priority: cell functions in the
environment or species identification?
• Different versions of cytometry to address quite different
situations (rare events, solid phase flow cytometry);
• Coupling cytometry and other techniques;
• Technical needs of experimenters.
14. Astrobiology ................................ Room 301
Chair: Sarah Baatout
This workshop is intended for scientists working or interested
in the field of space biology, especially as it relates to cytometric
analysis, and aims to further the development of appropriate
cytometric methods for the study of the effects of microgravity
on living organisms. The workshop will include presentations
from researchers applying contemporary cytometric
approaches to analyze the effects of space conditions,
microgravity, and/or radiation on cells and organisms. The
application of specific cytometric methods to the study cell
metabolism, apoptosis, DNA repair and in human and bacterial
cells cultured in space or microgravity conditions will be
discussed. Finally, modifications of cytometric instrumentation
necessary to make them compatible with use in space or in
the absence of microgravity will also be discussed.
Over the past few years, there have been several discussions
within the cytometry community regarding the determination
of receptor positivity and the appropriate use of isotype controls.
The goal of this workshop is to develop a plan that identifies
“what needs to be done” to resolve confusion surrounding the
use of isotype controls and the determination of receptor
positivity, with the aim of providing guidance for novice and
experienced cytometrists alike. The objectives of this plan
would include:
1. Preparation of a comprehensive consensus guideline for
determining background levels of fluorescence; 2. Suggestions
for the best model(s) for determining receptor positivity. 3.
Education of users and reviewers of the limitations of isotype
control use.
16. Flow Cytometric Analysis of ZAP-70 in BCLL: Are There Alternatives? ....... Room 302
Chair: Jan Phillipe
This workshop will focus on the technical issues in ZAP-70
measurement and its clinical applications, with the following
questions in mind: Is ZAP-70 analysis as prognostic marker in
B-CLL useful? What is the best choice for ZAP-70 analysis: flow
cytometry or molecular analysis? Are there alternatives? Will
these kinds of markers remain relevant in the future?
17. Laser Scanning Cytometry .......... Room 304
Chair: Attila Tarnock
Quantitative and stoichiometric analysis on the single cell level,
Slide Based Cytometry (SBC), has become an important
analytical technology in drug discovery and research and is an
emerging technology for clinical diagnosis. SBC enables to
perform high-content high-throughput analysis from cell
suspensions over cell cultures to tissues. In the last years a
great number of SBC instrumentation has been launched.
However, the technical realizations are very diverse with respect
to the technology applied (differences in: light sources,
detectors, color detection, optical paths, confocal or nonconfocal detection), the residual image formats, the image
analysis used to recognize single cells (cell detection modes)
and resulting data formats. Presently most of the instruments
are unique and standardization as well as comparability of
different instruments is a major challenge. The aim of this
workshop is to discuss and define with the participants the key
aspects for standardization of the different instruments in order
to enable intra-system quality assessment and for comparison
65
between systems.This also includes standardization of sample
handling data analysis and data output. We will collect the
most technical aspects from the majority of available
commercial instruments that need to be addressed. As a result
of this workshop we intend to produce a consensus statement
that will be made available to the users and the manufacturers.
18. Flow Cytometry Calibration and
Standardization ................... Room 205 B/C
in use for decades and are relatively non-controversial. Other
tasks, such as characterizing instrument performance in
absolute terms, or comparing measurements performed on
different instruments with different reagents involve many
issues and the best approach are still subjects of debate. As
one component of an effort by ISAC to help the community
reach consensus on the best approaches to the calibration and
standardization of flow cytometry instruments and
measurements, this workshop aims to identify areas of
consensus so that clear guidance can be provided to the novice,
as well as to discuss charting science-based paths to establish
consensus on more controversial issues.
The calibration and standardization of flow cytometry
instruments and measurements is critical to its use as a
quantitative tool to study biological systems. Some procedures,
such as using fluorescent microspheres to ensure that sample
stream, laser beam, and detectors are in alignment have been
Special Workshops
Human Cell Differentiation
Molecules ............................................. Room 304
Welcome/Introduction
Heddy Zola
14:10 – 14:30
Defining Subsets of Fibroblasts
Chris Buckley
14:30 – 14:50
Members of CD150 (SLAM) Family As
Human Cell Differentiation
Pablo Engel
14:50 – 15:10
CD300: A Family of Leukocyte Regulatory
Molecules
Georgina Clark
15:30 – 15:50
Intracellular Molecules for the Study of
Tissue Biopsies
David Mason
66
14:00 - 17:30
15:50 – 16:10
Phospho-specific Antibodies As Markers of
Differentiation
Bob Balderas
16:10 – 16:30
FoxP3 Antibodies As Markers of Regulatory
T Cells
Allison Banham
16:30 – 16:50
Time and Space Resolved Analysis of
Function of Leukocyte Antigens by Ultrasensitive Single Molecule Imaging
Hannes Stockinger
16:50 – 17:10
HCDM Workshop Results
• Regulatory T Cells
• Orphan Antibodies
• New CD Designations
Heddy Zola and Bernadette Swart
17:10 – 17:30
Closing Remarks from the President of
HCDM/HLDA
Laurence Boumsell
Please see abstracts 143-148.
14:00 – 14:10
Monday, 22 May
Tuesday, 23 May
Special Workshops
Resource Managers’ Workshop ........ Room 204A
19:00 – 19:15
Introduction: The Modern Resource
Manager: Educator, Administrator, Scientist
- Maintaining the Balance
Julie Auger
19:15 – 19:45
Crisis Management
Linda Lopez
19:45 – 20:15
Measuring Laboratory Output
Derek Davies
19:00 - 22:30
20:15 – 21:00
Electronic Tools for Lab Management
Wayne Green and Leonore Herzenberg
21:00 – 21:15
Break
21:15 – 21:30
ISAC Strategic Plan Overview
Lori Krueger
21:30 – 22:00
Education in a Resource Laboratory: One
Lab’s Experience
Jonni Moore
22:00 – 22:30
Ask the Experts Panel Discussion
Marie Follo
22:30
Adjourn
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Commercial Tutorials
Sunday, 21 May
New Lasers. New Optics. New BD
FACSDiva™Software. Innovations and developments at BD Biosciences for 2006.
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408 432-9475
Fax: 408 954-2009
www.bdbiosciences.com
12:00 – 13:00 – Room 301
Presenter: Joe Trotter
This tutorial will present information and data on new lasers and optics currently under development at BD Biosciences, an update on platform software development,
and cover useful operating procedures to guarantee optimal system performance, especially for multicolor assays.
Applications for Large Particle Flow Cytometry
Union Biometrica, Inc.
84 October Hill Road
Holliston, MA 01746
Phone: 508 893-3115
Fax: 508 893-8044
Email: [email protected]
www.unionbio.com
12:00 – 13:00, Room 204A
Presenter: Rico Bongaarts
Many objects are too large and fragile for conventional
flow cytometry. Manual microscopic manipulation of these
objects is tedious, subjective, and limits experiments size /
scope. In this tutorial, we discuss using large particle flow
cytometry for automating the process of analyzing and
sorting large (20-1,500 micron) particles at a rate of 10-30
objects/second. Using object size, optical density, and intensity of fluorescent markers as sorting criteria, selected
objects may be dispensed in bulk / multi-well plates. This
technique is ideal for use with live biological materials and
sensitive chemistries because a non-destructive pneumatic
68
sorting mechanism and lower shear forces (due to a slower
fluid stream) avoid harming or changing objects. Applications examples to be discussed include: large cells /cell
clusters (embryonic stem cells, pancreatic islets, hepatocytes); bead-based libraries; and model organisms
(C.elegans, Drosophila, Zebrafish).
Flow Cytometric Analysis of Signal Transduction
Networks
200 S. Kraemer Boulevard
Brea, CA 92821
Phone: 714 993-8723
Fax: 714 961-4826
www.beckmancoulter.com
13:00 – 14:00 – Room 302
Presenter: T. Vincent Shankey. Advanced Technology Center, Beckman Coulter, Inc.
Miami FL
Signal transduction pathways represent the major communications networks that transmit signals from multiple
cell surface receptors to the nucleus, generally resulting
in cell death, survival, proliferation or differentiation. Several limitations impact the measurement of signal transduction networks by flow cytometry, including difficulties
in producing antibodies to specific protein phosphorylation
epitopes and limitations imposed by cell fixation and
permeabilization. We have developed a technique to monitor phosphorylation-specific epitopes in tissue culture cells,
and have extended these studies to the analysis of phosphoepitopes in clinical samples, including whole blood and bone
marrow aspirates. We are using flow cytometry to monitor signal transduction pathways in human leukemias, including the impact of Gleevec™ on phosphorylation of
STAT5 in Chronic Myelogenous Leukemia, key pathways
in Acute Myelogenous Leukemia, including P-ERK and
the PI3 kinase pathways (measuring P-AKT and P-S6),
and measurement of ZAP-70 protein expression in Chronic
Lymphocytic Leukemia. These studies provide important
insight into the underlying biology, and provide potential
biomarkers for monitoring the impact of targeted inhibitors of signaling pathways in clinical samples using flow
cytometry.
Use Of Qdot® Nanocrystals for Imaging and Flow
Cytometry Applications
Invitrogen
29851 Willow Creek Road
Eugene, OR 97402-9132
Phone: 541 465-8300
Fax: 541 335-0305
www.probes.invitrogen.com
13:00 – 14:00 – Room 202
Presenter: William Godfrey, Ph.D. Flow
Cytometry Research Area Manager
Quantum dots are nanometer-scale particles whose composition and small size (hundreds to thousands of atoms)
give them extraordinary optical properties. Their most striking property is that the color of quantum dots can be “tuned”
(quantum confinement effect). By using relatively few
semiconductor materials and an array of sizes, quantum
dots can be prepared with fluorescence properties spanning the spectrum from the ultraviolet to the infrared.
Quantum dot emission is exceptionally narrow and symmetric, leading to easy multiplexing with minimal need to
compensate for spectral overlap. Available Qdot ®
nanocrystals are readily excited by UV through blue wavelengths, are brightly fluorescent, and are exceptionally
photo-inert. Their unique polymer-based surface coating
allows Qdot nanocrystals to be readily conjugated to a
variety of ligands, including antibodies, peptides, oligos, and
organic molecules. These reagents can be used in virtually the same range of applications as organic fluors, including immunohistology, immunophenotyping, cell tracking, FISH, microarray detection, and in vivo imaging.
Imaging and flow cytometry examples will be presented.
Accessing Dynamic Parameters within Cells by
Combining Imaging and Fluorescence Fluctuation
Evotec Technologies GmbH
Schnackenburgallee 114
D-22525 Hamburg
Germany
We have combined fluorescence imaging with fluctuation
based single molecule detection to analyze single living
cells. Using the same positioning and detection system for
laser scanning and point fluctuation acquisition, the following parameters within cells (e.g. from GFP labeled
proteins) can be accessed:
1. Absolute concentrations, 2. Lateral and 3. Rotational
diffusion times, 4. Fluorescence quench (molecular intensity), 5. Fluorescence lifetime, 6. Energy transfer (FRET),
7. Multimerisation, 8. Aggregation and 9. Coincidence.
As an example, we present measurements of the membrane potential detection in adherent CHO cells using the
advantages of image processing and photon statistics
methods FIDA and FIDA-2D. Using dual color coincidence information the membrane potential across a CHO
plasma membrane can be determined.
Monday, 22 May
Every Dot Has a Story: Multispectral Image
Analysis of Cells in Flow with the Amnis
ImageStream System
Amnis Corporation
2505 3rd Avenue, Suite 210
Seattle, WA 98121
Phone: 206 576-6890
Fax: 206 576-6895
www.amnis.com
12:00 – 14:00 – Room 205 B/C
Presenter: David Basiji, Ph.D.
The advent of high resolution, multi-spectral image analysis of cells in flow has enabled a wide range of new applications. In this tutorial, Amnis scientists and ImageStream
users discuss the work they’ve been doing with this new
technology and ways it has been applied to enhance the
capabilities of both fluorescence microscopy and conventional flow cytometry.
www.evotec-technologies.com
13:00 – 14:00 - Room 303
Presenter: Dr. Stefan Jäger
The need for more specific information on a molecular level
deduced from in vivo cell assays has grown since novel
molecular imaging technologies have been established.
69
Standards Rule! A Program for Standardized
Instrument QC, Set-Up and Quantitative Fluorescence Measurements
Bangs Laboratories, Inc.
9025 Technology Drive
Fishers, IN 46038
Phone: 317 570-7020, 800 387-0672
Fax: 317-570-7034
www.bangslabs.com
12:00 – 13:00 – Room 204A
Presenter: Kathy Turner, Bangs Laboratories
In the field, service engineers rely on microsphere standards to check and calibrate flow cytometers. Similar
standards should be used by the laboratory as part of a
comprehensive quality control program. Microsphere standards aid in defining cytometer capabilities and limitations
in terms of sensitivity, precision and accuracy, and provide a means for ensuring that the instrument is stable and
suitable for use. They are also helpful in understanding
the effects of extraneous factors such as temperature,
humidity, and electronic noise. Furthermore, microsphere
standards have become imperative for applications in fluorescence quantitation.
This tutorial will outline a basic program for routine quality control and standardization. Topics will include:
• Understanding instrument capabilities
• Daily Instrument QC
• Daily Instrument set-up—defining the “Window of
Analysis”
• Quantitative fluorescence cytometry—standardized fluorescence intensity measurements
Cytometer Setup and Tracking in a Multi-color,
Digital World
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408 432-9475
Fax: 408 954-2009
12:00 – 13:00 – Room 301
Presenter: Alan Stall, Ph.D.
To take full advantage of modern flow cytometers capable of measuring up to 12-16 individual digital fluorescence parameters it is critical that they are properly setup,
optimized and standardized for day-to-day performance
70
and reproducibility. This presentation focuses on the theory
and practice of measuring and optimizing cytometer performance including 1) optimization of laser delays; 2) determining linearity of fluorescence signals and how nonlinearity can affect compensation; 3) the difference between threshold and resolution sensitivity and how to optimize the latter and 4) daily standardization of PMT voltages / MFIs. Finally, a strategy for automating daily Cytometer Set-up and Tracking utilizing these concepts and
practices will be demonstrated.
High Content Imaging: Taking Cell Analysis to a
Higher Resolution
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408 432-9475
Fax: 408 954-2009
12:00 – 13:00 – Room 302
Presenter: David Cheo, Principal Scientist,
Bioimaging Systems
High content imaging is rapidly becoming a mainstay in
pharmaceutical and life-science research laboratories. The
power of imaging resides in its ability to measure not only
fluorescence intensity changes within cells, but also molecular redistribution and morphological features of cells
and tissues in a 3-D environment. In recent years, the
availability of a new generation of automated high content
imaging platforms has created a unique opportunity to
develop novel cell-based assays. To be used effectively in
cell analysis, an automated imaging system must provide
a wide range of capabilities including live-cell kinetic imaging, endpoint imaging and high-resolution confocal imaging. These features of the BD Pathway Bioimager,
coupled with versatile user configurable image data acquisition, processing and analysis software, allow the researcher to make multi-parameter measurements of cellular events in multi-dimensional space and time. This presentation will highlight a number of specific applications
developed for the BD Pathway system that demonstrate
its effectiveness as a powerful analytical cell biology research tool.
Sorting In a Parallel Universe: N-Flow Channel
Systems Enable Exploration of Bold New Frontiers, Going Where no Sorter has Gone Before
iCyt Mission Technology
P.O. Box 1593
Champaign, IL 61824-1593
Phone: 217 328-9396
Fax: 217 328-9692
www.i-cyt.com
12:00 – 13:00 – Room 205A
Presenter: Gary Durack
Some time ago, a customer posed a difficult question to
the professional engineering team at iCyt: “Is it possible
to create a cell sorter with throughput exceeding 2.5 x
109 cells per hour per operator, with reliable 24/7
operation, a high degree of automation, and perhaps
most importantly, an affordable cost?” iCyt answered
that question resoundingly by creating a multi-flow-channel instrument based on the revolutionary Highly Automated Parallel Sorting (HAPS) technology platform.
Employing 100% digital systems, the iCyt instrument is
capable of automated start-up and shut-down, automated
optical alignment, automated sort calibration, automated
setup of droplet break-off and continuous control, sort
population tracking with automated region setup, and continuous monitoring and fault correction. Envision the productivity improvement achieved by multiple, simultaneous
sorts. Imagine the reality of a sizeable bulk sort taking 2
hours instead of 10. Embrace the possibilities of serial
chain sorting where enrichment sorts automatically feed
purity sorts – all in a fraction of the time previously required. Welcome to the world of sorting in a parallel universe – where N-flow channel systems enable exploration of bold new frontiers!
Maximizing Use of the Violet Laser
Invitrogen
Eugene, OR 97402-9132
Phone: 541 465-8300
Fax: 541 335-0305
12:00 – 13:00 – Room 202
Presenter: Gayle Buller, MT (ASCP), R&D
Scientist
Solid state lasers, such as 405nm or 407nm violet diodes,
are becoming more prevalent in the next generation flow
cytometers because of their small size, low power requirements, cost effectiveness, and reliability over time. Widespread use of these lasers has been limited by availability
of fluorescent reagents excited by the violet laser.
Invitrogen provides a broad range of violet-excitable tools
to unlock the full potential of flow cytometer equipped
with a violet laser. This seminar will present novel fluorescent dyes and reagents that are designed for optimal
utilization of the violet laser. Among the areas to be discussed are multicolored immumophenotyping (including the
use of QDot nanocrystals), viability and vitality reagents,
and cell cycle analysis.
Cell Cycle Analysis in Live Cells
Invitrogen
Eugene, OR 97402-9132
Phone: 541 465-8300
Fax: 541 335-0305
13:00 – 14:00 – Room 202
Presenter: Jolene A. Bradford, MT (ASCP), CLS
(NCA), R&D Scientist
Live cell studies of cellular DNA content and cell cycle
distribution are useful to detect variations of growth patterns due to a variety of physical, chemical, or biological
means, to monitor apoptosis, and to study tumor behavior
and suppressor gene mechanisms. Classically performed
on fixed or permealibized cells using a cell-impermeant
nucleic acid stain, cell cycle analysis using a cell-permeant
dye is ideally suited for simultaneous detection of cell cycle
phase, with other live cell function testing such as
immunophenotyping and/or apoptotic evaluation. This tutorial will focus on unique fluorescent reagents currently
71
available to analyze cell cycle progression in living cells.
Examples of live cell staining for DNA content will be
presented. Technical issues and gating strategies will be
discussed. In addition, applications for cell cycle analysis
using live cell staining will be presented including apoptosis
studies and sorting based on DNA content.
FlowJo Basics 101: Learn Mo’ about the ‘Jo
Tree Star, Inc
340 A Street #203
Ashland, OR 97520
Phone: 800 366-6045
Fax: 541 482-3153
www.flowjo.com
12:00 – 13:00 – Room 304
Presenter: Jennifer Wilshire, Ph.D.
Perfect for Beginner FlowJocks... FlowJo is an analysis
software with extensive tools for immunophenotyping
analyses. FlowJo can read data files acquired on ANY
flow cytometer and runs on both Mac and PC computers.
This presentation demonstrates how to analyze
immunophenotyping data efficiently and easily with
FlowJo. We’ll start with raw data files and finish by generating summaries of the entire experiment (tables and
graphical reports). FlowJo’s batching function creates
these reports with one click of a button. The FlowJo software is intuitive and easy to learn!
Use of Activation State-Specific Antibodies in
Flow and Imaging Cytometric Applications
Cell Signaling Technology
3 Trask Lane
Danvers, MA 01923
Phone: 978 867-2300
Fax: 978 867-2400
www.cellsignal.com
12:30 – 13:30 – Room 303
Presenter: Randy Wetzel
As therapeutics and diagnostics become more focused on
the underlying mechanisms of disease, cellular analysis of
intracellular signaling proteins will be increasingly important. Modifications such as phosphorylation, cleavage, and
acetylation are central regulatory mechanisms of intracellular proteins. Therefore, it is necessary to examine not
only expression and localization of signaling proteins in
cells and tissues, but also to determine their activity level.
72
Activation state-specific antibodies can be easily combined
with cytometric methods to examine abnormal signaling
associated with cancer and other diseases. They can also
be used to study signaling events associated with cell cycle,
apoptosis, stem cell maintenance and differentiation, inflammation, or DNA damage. This tutorial will describe
methods for the analysis of cellular signaling using flow
cytometry, immunofluorescence, and plate-based high
throughput/high content screening applications. We will
also discuss the latest fixation/permeabilization protocols
that allow the concurrent use of signaling antibodies with
surface markers and other dyes in flow cytometry.
Software Solution for Bead-Based Immunoassays
Soft Flow Hungary Ltd.
20 Kedves St
Pecs, H-7628
Hungary
Phone: +36 72 240064
Fax: +36 72 240065
www.softflow.com
13:00 – 14:00 – Room 204B
Presenter: Gyorgy Lustyik
The tutorial will provide an overview of the new FCAP
Array software, a powerful, scientific analysis package
designed for Cytometric Bead Array (CBA) data. The
software supports the flexible, open-architecture design
of most commercially available microbead based technologies. With FCAP Array, these technologies can be configured to perform a wide variety of bioassays quickly and
ac curately. Workflow topics covered will include the creation and data processing of immunoassays performed with
BDT FlexSet cell signaling reagents on a caveolae biological model using HepG2 liver carcinoma cell line.
Caveolae are specialized plasma membrane domains that
play a significant role in various human patho-biological
conditions such as some cardiovascular diseases, cancer,
diabetes, muscular dystrophy, and in the pathogenesis of
various additional human diseases. Data acquired with
several commercial flow instruments will be presented.
The adata analysis will be demonstrated on both version
of FCAP Array running in Microsoft Windows® and Apple
Mac OS® operating systems.
Tuesday, 23 May
Lyoplate Technology: A New Paradigm for Flow
Based Assay Design and Implementation
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 408 432-9475
Fax: 408 954-2009
12:00 – 13:00 – Room 301
Presenter: John Dunne, Ph.D., Associate Scientific
Director
The complexity of the human immune system can be explored by analysing lymphocyte cytokine responses to
panels of specific antigens. Flow cytometry enables detailed description of such responses, and with recent technical developments including lyophilized reagents in 96well plates and automatic gating tools, it can be used to
map such responses against normal chronic infections like
CMV and idiosyncratic responses to tumor antigens in
asymptomatic donors. These studies can help anticipate
heterogeneity in normal donors, an important component
in measuring changes related to immunomodulatory therapies.
cGMP Sorting of Hematopoietic Stem Cells
Dako
6392 Via Real
Carpinteria, CA 93013
Phone: 800 235-5743
Fax: 805 566-6688
www.dako.com
12:00 – 13:00 – Room 302
Presenter: Brenda R. Lee
CD34+ cells from G-CSF mobilized blood are sorted using
a modified Dako MoFlo® to generate quantities of HSC
required for transplantation for hematpoietic reconstitution. Modifications made to the MoFlo® allow aseptic
conditions to be maintained during sorting. Methyl cellulose, LTC-IC Assays and xeno-engraftment of HSCs using the NOD-SCID mouse model are used to demonstrate
functionality of the MoFlo® sorted HSC.
Immunophenotypic Analysis of CMV-Specific T
Cells Using MHC Dextramer Technology
Dako
6392 Via Real
Phone: 800 235-5743
Fax: 805 566-6688
www.dako.com
13:00 – 14:00 – Room 302
Presenter: Jan A. Bayer, Ph.D.
Using MHC Dextramers, we studied CMV-specific T cell
populations with the purpose of obtaining detailed information on their immunophenotype, as well as on the usage of V²-chains in CMV-specific T cells. For that purpose we applied 9-color analysis to a cohort of HLA-typed
healthy donors with positive CMV serological status. For
individuals presenting multiple candidate HLA alleles, each
allele was evaluated using the appropriate multimers.
FlowJo Advanced: New Features and Advanced
Topics
Tree Star, Inc
340 A Street #203
Ashland, OR 97520
Phone: 800 366-6045
Fax: 541 482-3153
www.flowjo.com
12:00 – 13:00 – Room 304
Presenter: Jennifer Wilshire, PhD
Feel like a FlowJo Wiz? Everyone can benefit from this
presentation. Learn tips and tricks that can help you analyze your data more efficiently. We will be demonstrating
advanced FlowJo topics such as... compensation, cell
cycle, proliferation. In addition we’ll show how easy it is
to set up templates for doing repetitive analyses of complex experiments . New versions of FlowJo have recently
been released for both Mac and PC and we’ll demonstrate some of the many new features.
73
A System for Automated Isolation of Single Cell
Clones
Evotec Technologies
Hamburg 22525
Germany
Phone: +49 40 560810
Fax: +49 40 56081488
www.evotec-technologies.com
13:00 – 14:00 – Room 303
Presenter: Dr. Torsten Mueller
Conventional methods for identification and isolation of
clonal cells are based on limited dilution or FACS sorting.
The single cell status has to be controlled by microscopy
or it is verified retrospectively. This process is tedious and
time-consuming. Our automated system employing advanced cell imaging and microfluidics technology is dedicated to live single cell isolation and makes the cloning
process very easy. It provides high-content information
during the cell isolation process. Suspended cells are analyzed by phase contrast and fluorescence microscopy, and
they are sorted using dielectrophoretic forces. Cells can
be selected according to viability, presence or absence of
fluorescence and size. High resolution images using up to
eight different fluorescence filter sets are obtained from
target cells of interest which are then deposited into a
micro titer plate. A selection according to image analysis
parameters is feasible. This unique combination of cell
imaging and gentle live cell isolation in a single step enables to pursue novel strategies for protein expression
analysis. Examples are given using various cell types
(CHO, U2OS, human neural progenitor cells, mouse embryonic stem cells).
74
Routine Detection and Quantitation of
Fetomaternal Hemorrhage Using Fetall Cell
Count™
IQ Products
Rozenburglaan 13a
Groningen 9727 DL
The Netherlands
Phone: +31 50 57 57 000
Fax: +31 50 57 57 002
www.iqproducts.nl
13:00 – 14:00 – Room 204A
Presenter: Joost Schuitemaker
Quantitative fetal red blood cell (fRBCs) detection in
maternal blood is important in obstetrical management as
it is used to estimate the extent of Fetomaternal Hemorrhage (FMH) during pregnancy. Possible causes are threatened miscarriage, trauma cases with suspected placental
injury or RhD incompatibility between fetus and mother
(Hemolytic Disease of the Newborn). Flow cytometry
(FCM) is an accurate, sensitive and reliable method for
FMH quantification. The Fetal Cell Count™ kit (FCC)
offers detection of two antigens. Fetal hemoglobin is highly
expressed in fetal RBCs and in low amount in adult RBCs.
Carbonic Anhydrase is present only in adult RBCs. Using
these markers a clear separated population of fRBCs is
quantitated. Clinical evaluation concluded that the FCC
kit can advantageously replace the manual microscopic
slide based Kleihauer-Betke test. Visual inspection of the
FCM pattern alone is sufficient to determine any present
fetal cells. The percentage cells counted in the UL quadrant area further precises the FMH amount showing its
usefulness of adapting the therapeutic treatment by aiding
in anti-RhD dose adjustments.
Dyes, Assays and Workflow Solutions for High
Throughput Imaging (Hcs/Hti)
PhycoLink® Conjugation and Purification Kits:
How to Make the Best Darn Conjugates
Invitrogen
Eugene, OR 97402-9132
Phone: 541 465-8300
Fax: 541 335-0305
ProZyme, Inc.
1933 Davis Street, Suite 207
San Leandro, CA 94577-1258
Phone: 510 638-6900; 800 457-9444
Fax: 510 638-6919
www.prozyme.com
13:00 – 14:00 – Room 202
13:00 – 14:00 – Room 205 B/C
Presenter: Michael J. Ignatius, Ph.D. Director Cell
Biology and Imaging
Presenter: Pat Burroughs
Molecular Probes/Invitrogen, has a diverse offering of low
cost reagents and assays. This presentation will detail
progress to codify and industrialize (to automate, standarize
and scale) our most useful dyes for low cost assays for
HCS/HTI type discovery efforts. Initial content will be
around the fundamentals: improved cell counting, viability,
vitality, apoptosis, calcium flux, nuclear markers and segmentation. Next, our new approaches to expression reporter read outs, from fluorescent proteins to betalactamase and Flash/Reash. Examples of toxicity profiling will be given, highlighting new assays for defects in
lipid loading/clearing (phospholipidosis and steatosis), mitochondrial stress and more. Finally in-progress efforts in
nanocrystals, following our recent acquisition of Quantum
Dot Corporation will be discussed. Most of these approaches are non-antibody based, suitable for mulitplexing
and many live-cell compatible, offering the potential to
affordably understand at a cellular level, complex physiological responses to thousands of test compounds.
Researchers increasingly conjugate their own antibodies
because they want direct conjugates; have only limited
quantities to work with; can’t find the right color on the
desired marker; want to reserve the brightest tags for their
dimmest antigens; or are driven by the need for more costeffective reagents. Others may just want to do it themselves or understand the basic principles. We’ve got the
answers for all of you. Please join us for a tutorial on
conjugating PE, APC, PerCP and other phycoproteins (with
subsequent conjugate purification) using ProZyme’s fast
and easy kits. We will also focus on those factors that
make conjugates bright, reducing the scale (50 ug or less),
evaluating conjugates for consistency lot-to-lot, scaling up
and troubleshooting. Get the benefit of years of experience in one short hour from the people who know
phycobiliproteins. PhycoLink is a registered trademark
of ProZyme, Inc.
Title not available prior to press time.
11800 S.W. 147th Avenue
Miami, FL 33196-2500
Phone: 800 526-3821
Fax: 800 232-3828
www.beckmancoulter.com
13:00 – 14:00, Room 204B
75
Exhibitor Listing
Company .................................................... Booth(s)
AbD Serotec ....................................................................... 208
Advalytix AG ...................................................................... 401
ALEXIS® Biochemicals ................................................... 316
Amnis Corporation ............................................... 510, 512
AnaSpec Inc. ...................................................................... 302
Apogee Flow Systems ................................................... 525
Asahi Spectra U.S.A. Inc. ................................................ 207
Bangs Laboratories, Inc. ................................................. 209
Bay bioscience Co., Ltd. ................................................. 312
BD Bioscience ............................. 225, 227, 229, 231,324,
.............................................................................. 326, 328,330
Beckman Coulter, Inc. .................................................... 700
BioLegend .......................................................................... 501
Biomedical Photometrics Inc./GeneFocus ........... 221
Biostatus Limited ............................................................ 624
Biotrue Inc. ......................................................................... 307
Brightwell Technologies Inc. ....................................... 602
Cedarlane Laboratories Limited ............................... 407
Cell Signaling Technology ............................................ 507
CellSeed Inc. ...................................................................... 628
Chemicon/Upstate ......................................................... 523
Chroma Technology Corp ............................................ 600
Cobolt AB ............................................................................ 405
Coherent Inc. ........................................................... 411, 413
CompuCyte Corporation ................................... 604, 606
CRI ......................................................................................... 500
Cyntellect, Inc. .................................................................. 309
CyTecs GmbH .......................................................... 422, 424
Cytek Development, Inc. .................................... 303, 305
CYTOPEIA, INC. ....................................................... 520, 522
Dako ...................................................................................... 800
De Novo Software ........................................................... 210
Duke Scientific Corp. ...................................................... 421
Evotec Technologies ....................................................... 408
Fluid Imaging Technologies, Inc. ............................... 423
Fraen Corporation Srl ..................................................... 310
Fujitsu Computer Systems........................................... 306
Company .................................................... Booth(s)
GCAT, Inc..................................................................... 325, 420
GE Healthcare ................................................................... 425
Guava Technologies, Inc. ..................................... 506, 508
iCyt Mission Technology ........................... 101, 103, 105,
............................................................................. 200, 202, 204
Immunicon Corporation .............................................. 409
Innovative Cell Technologies, Inc. ............................. 214
Invitrogen ................................................................. 415, 417
IQ Products ......................................................................... 308
ISAC Booth .......................................................................... 311
Jackson ImmunoResearch Laboratories ................ 514
JDSU ...................................................................................... 314
LightForm, Inc. .................................................................. 205
MAIA Scientific ................................................................. 320
Miltenyi Biotec ....................................................... 516, 518
Molecular Devices .......................................................... 218
MSP Corporation ............................................................. 402
New Mexico Molecular Libraries
Screening Center ........................................................ 403
Newport Corporation .................................................... 404
Nikon Instruments Inc. .................................................. 211
Omega Optical, Inc. ........................................................ 300
Parker Hannifin - Pneutronics Division ................... 203
Partec GmbH ........................................................... 321, 323
Phoenix Flow Systems, Inc. .......................................... 212
Picarro .................................................................................. 201
ProZyme, Inc. ..................................................................... 219
Qimaging ............................................................................ 109
ScienceXperts .................................................................. 528
Soft Flow Hungary Ltd. ................................................... 406
SouthernBiotech .............................................................. 419
Spectral Applied Research .......................................... 504
Spherotech Inc. ................................................................ 622
Tree Star, Inc. ...................................................................... 301
TTP LabTech Ltd. .............................................................. 318
Union Biometrica, Inc. ................................................... 304
Verity Software House ......................................... 427, 429
Wiley ..................................................................................... 400
Exhibit Hours
Sunday, 21 May, 12:00 – 16:00 and 19:00 – 21:00 (Opening Reception in hall)
Monday, 22 May, 9:30 – 14:00 and 15:30 – 19:30 (Happy Hour in hall)
Tuesday, 23 May, 9:30 – 14:00 and 15:30 – 19:30 (Happy Hour in hall)
Wednesday, 24 May, 9:30 – 12:00
76
As of 17 April
Exhibitor Floorplan
77
Exhibits
AbD Serotec .......................................................... 208
3200 Atlantic Avenue, Suite 105
Raleigh, NC 27604
Phone: 800 265-7376
Fax: 919 878-3751
Web: http://www.serotec.com
AbD Serotec, a division of MorphoSys, is one of the world’s
leading producers of immunologicals. Founded in 1982 in
response to demand for high quality antibodies, AbD
Serotec’s thousands of reagents include antibodies for
human, rodent and veterinary research useful in the study
of immunology, neurology, cell biology and histology.
Advalytix AG ......................................................... 401
58 Elsinore Street
Concord, MA 01742
Phone: 978 405-2533
Fax: 978 405-2534
Web: http://www.advalytix.com
Advalytix, an Olympus subsidiary, is introducing the
AmpliGrid 1µl PCR slide for affordable, high-throughput
genetic analysis of cells pre-sorted by immunologic and
morphologic characteristics.
Cells are sorted into the slide’s 48 reaction sites where lysing and amplification are performed - no reformatting. Available automation allows reliable identification of rare cells.
Amnis Corporation ...................................... 510, 512
2505 Third Avenue, Suite 210
Seattle, WA 98121
Phone: 206 576-6890
Fax: 206 576-6895
Web: http://www.amnis.com
AnaSpec Inc. .......................................................... 302
2149 O’Toole Avenue
San Jose, CA 95131
Phone: 408 452-5055
Fax: 408 452-5059
Web: http://www.anaspec.com
AnaSpec, Inc. is a leading provider of integrated proteomics
solutions for worldwide life science research. With a vision for innovation through synergy, AnaSpec offers expertise in three primary technologies: peptides, detection
reagents, and combinatorial chemistry.
78
Apogee Flow Systems .......................................... 525
Enterprise House, Maxted Road
Hemel Hempstead
Herts, HP2 7BT
U.K.
Phone: 44 207 0434 137
Fax: 44 1442 231098
Web: http://www.ApogeeFlow.com
Apogee Flow Systems manufacture flow cytometers suitable for both benchtop and field use. The Apogee A40
system is optimized for superb performance in detection
of bacteria, large viruses or other micro-particles. A general purpose version is also available. Apogee is currently
seeking distributors and partners with whom to expand its
business.
Asahi Spectra U.S.A. Inc. .................................... 207
23505 Crenshaw Boulevard, Suite 225
Torrance, CA 90505
Phone: 310 530-5855
Fax: 310 325-8974
Web: http://www.asahi-spectra.com
Alexis Biochemicals ............................................. 316
6181 Cornerstone Court East, Suite 103
San Diego, CA 92121
Phone: 800 900-0065
Fax: 800 550-8825
Web: http://www.axxora.com
ALEXIS® Biochemicals produces innivative life science
reagents. Featured product groups include the latest
biochemicals, immunochemicals, fluorescent tools and
assay kits for research involving Apoptosis, Cancer CellCycle, Gene Regulation, Immunology, Inflammation, Nitric Oxide, and Signal Transduction. Visit
www.axxora.com, your direct link to original manufacturers.
Bangs Laboratories, Inc. ..................................... 209
9025 Technology Drive
Fishers, IN 46038-2886
Phone: 317 570-7020
Fax: 317 570-7034
Web: http://www.bangslabs.com
Beckman Coulter, Inc. ......................................... 700
4300 N. Harbor Boulevard
Fullerton, CA 92834-3100
Phone: 714 871-4848
Fax: 714 773-8283
Web: http://www.beckmancoulter.com
Bangs Laboratories produces fluorescence microsphere
standards to help the research and clinical community conduct studies and evaluate data in an easier, more precise
manner. Our products set the standard for quality control
and quantitation in the fields of flow cytometry, fluorescence microscopy, image analysis, and other fluorescence
analytical applications.
Beckman Coulter, Inc. is a leading manufacturer of biomedical testing instrument systems, tests and supplies that
simplify and automate laboratory processes. Spanning the
biomedical testing continuum—from pioneering medical
research and clinical trials to laboratory diagnostics and
point-of-care testing—Beckman Coulter’s systems provide essential biomedical information to enhance health
care around the world.
Bay Bioscience Co., Ltd. ..................................... 312
5-5-2-505, Minatojima Minamimachi,
Chuo-ku Kobe 650-0047, Japan
Phone: 81 78 304 5881
Fax: 81 78 304 5889
Web: http://www.baybio.co.jp
BioLegend ............................................................. 501
8395 Camino Santa Fe, Suite E
San Diego, CA 92121
Phone: 858 455-9588
Fax: 858 455-9587
Web: http://www.biolegend.com
JSAN, the new generation desktop cell sorter developed
with a grant from the Japanese government, requires minimal laser and sort setting adjustment. This enables researchers to analyze and sort cells more easily and accurately without a highly experienced operator. JSAN has
two models: a 2-LASER (488nm/638nm) 6-color model
and a 3-LASER(375nm or 405nm) 8-color model.
BioLegend manufactures reagents for immunology, cell
biology, and cancer research. BioLegend’s broad offering of antibody conjugates, including tandem dyes, Alexa
Fluor®, and Pacific Blue™, provides unsurpassed flexibility for multi-color flow analysis. Novel antibodies:
FOXP3, CCR7, IL-23(p19), PARC, TREM-1, NOD2.
BioLegend offers antibodies for ELISA, ELISPOT, immunohistochemistry, and Western blotting applications.
BD Bioscience ...........................225, 227, 229, 231,
....................................................... 330, 324, 326, 329
2350 Qume Drive
San Jose, CA 95131
Phone: 408 954-2296
Fax: 408 621-4227
Web: http://www.bdbiosciences.com
BD, a leading global medical technology company that
makes and sells medical devices, instrumented systems
and reagents, is dedicated to improving people’s health
throughout the world. BD is focused on improving drug
therapy, enhancing the quality and speed of diagnosing
infectious diseases, and advancing research and discovery of new drugs and vaccines. The Company serves
healthcare institutions, life science researchers, clinical
laboratories, industry and the general public.
Biomedical Photometrics Inc./GeneFocus ....... 221
550 Parkside Drive, Unit A12 Waterloo,
ON N2L 5V4, Canada
Phone: 519 886-9013
Fax: 519 886-5300
Web: http://www.confocal.com
The TISSUEscope™, awarded Frost & Sullivan’s 2005
Technology Innovation of the Year Award in Confocal
Imaging Solutions, is based on BPI’s patented
MACROscope® confocal laser scanning technology. The
TISSUEscope™ is an integrated scanning system for fluorescent, brightfield and reflected light digital imaging of
tissue and tissue microarrays at submicron resolution.
79
Biostatus Limited ................................................. 624
56 Charnwood Road,
Shepshed Leicestershire LE12 9NP, U.K.
Phone: 44 1509 558 163
Fax: 44 1509 651 061
Web: http://www.biostatus.co.uk
Cedarlane Laboratories Limited ........................ 407
5516-8th Line, RR#2
Hornby, ON L0P 1E0, Canada
Phone: 905 878-8891
Fax: 905 878-7800
Web: http://www.edarlanelabs.com
Biostatus make novel reagents for cell-based science. Our
first product, DRAQ5™ is a LIVE cell DNA dye with
emission in the far-red. APOPTRAK™ has the same
emission profile but allows the positive discrimination of
LIVE and apoptotic cells. Our newest product, CyGEL™
links flow cytometry to microscopical analysis.
CEDARLANE® Laboratories Limited is a leading supplier of high quality reagents to the research and diagnostic communities. We specialize in providing antibodies in
various formats to mouse, rat and human specificities.
Other offerings include Cedarlane’s Lympholyte® cell
separation media, cell purification Immunocolumns,
Complement and Custom Antibody Services. Cedarlane
also distributes for over 250 companies within Canada.
Biotrue Inc. ............................................................ 307
575 Market Street, Suite 2475
San Francisco, CA 94105
Phone: 415 438-2662
Fax: 415 276-4759
Web: http://www.biotrue.net
Biotrue develops software systems that enable biomedical researchers to easily store, manage and share all types
of instrument and analytical datafiles. Biotrue’s solutions
simplify search and retrieval, facilitate research collaboration and reduce the risk of data loss, all through a web
browser, so you can focus on research rather than worry
about data.
Brightwell Technologies Inc. .............................. 602
195 Stafford Road, West Ottawa,
ON K2H 9C1, Canada
Phone: 613 829-2772
Fax: 613 829-4921
Web: http://www.brightwelltech.com
Brightwell Technologies Inc. designs and manufactures
cell population analysis instrumentation utilizing Micro-Flow
Imaging™ technology. MFI™ Instrumentation provides
rapid measurement of size, shape, concentration & transparency of liquid borne cells through automated image
acquisition and analysis. Analyze discreet samples or
monitor continuously in real time!
80
CellSeed Inc. ......................................................... 628
R-Bldg. Shinjuku 1F, 33-8, Wakamatsu-cho,
Shinjuku-ku
Tokyo, 162-0056, Japan
Phone: 81 3 5286-6231
Fax: 81 3 5286-6233
Web: http://www.cellseed.com
CellSeed is a biotechnology innovator focused on novel
surface and cell culture products. It is dedicated to providing innovative solutions for tissue-engineered medicines
as well as progressive application of the technology background to basic medical drug discovery research. CellSeed’s
cell culture products are now commercially available for
laboratory use.
Cell Signaling Technology ................................... 507
3 Trask Lane
Danvers, MA 01923
Phone: 978 867-2300
Fax: 978 867-2400
Web: http://www.cellsignal.com
Cell Signaling Technology, Inc. is dedicated to providing
innovative high-quality activation state-specific antibodies
validated and formulated for use with flow cytometry,
immunofluorescence, high content analysis and other
cytometric applications. CST’s phospho-specific, cleavage-specific, and kinase motif antibodies enable clinicians
and researchers to examine abnormal signaling patterns
associated with disease, evaluate therapeutic efficacy, or
identify biomarkers and potential therapeutic targets.
Chemicon/Upstate ................................................ 523
28820 Single Oak Drive
Temecula, CA 92590
Phone: 951 676-8080
Fax: 951 506-0942
Web: http://www.chemicon.com
Chemicon International and Upstate are subsidiaries of
Serologicals Corporation, a global provider of biological
products and enabling technologies for research and cell
culture. We serve a variety of research needs in the areas of cell signaling, neuroscience, apoptosis, matrix biology and stem cell biology.
Chroma Technology Corp. .................................. 600
10 Imtec Lane, PO Box 489
Rockingham, VT 05101
Phone: 802 428-2500
Fax: 802 428-2525
Web: http://www.chroma.com
Chroma specializes in the design and manufacture of precision optical filters and coatings. We provide the greatest
accuracy in color separation, optical quality and signal
purity for applications such as low-light fluorescence microscopy and cytometry; spectrographic imaging in optical microscopy; laser-based confocal and multi-photon
instrumentation; and Raman spectroscopy.
Cobolt AB .............................................................. 405
Kraftiket 8
Stockholm SE-10405, Sweden
Phone: 46 8 5491230
Fax: 46 8 54591231
Web: www.cobolt.se
Cobolt supplies CW diode-pumped solid-state lasers
(DPSSLs) in the visible range, suitable for flow cytometry,
microscopy and sequencing applications. Cobolt lasers
combine high power and wavelength flexibility with low
noise and compact size.
Coherent Inc. ............................................... 411, 413
5100 Patrick Henry Drive
Santa Clara, CA 95054
Phone: 408 764-4983
Fax: 408 764-4646
Web: http://www.coherent.com
CompuCyte Corporation ............................ 604, 606
12 Emily Street
Cambridge, MA 02139
Phone: 617 577-4500
Fax: 617 577-4501
Web: http://www.compucyte.com
CRi .......................................................................... 500
35B Cabot Road
Woburn, MA 01801
Phone: 781 935-9099
Fax: 781 935-3388
Web: http://www.cri-inc.com
The CRi Nuance™ system’s unique hardware and algorithms enable multiplexing of biomarkers in both brightfield
and fluorescence. This can be used to image and separate multiple chromogens in immunohistochemistry or to
remove autofluorescence in difficult tissue sample. It can
also be used to enhance legibility in difficult FISH specimens.
Cyntellect, Inc. ...................................................... 309
6199 Cornerstone Court, Suite 111
San Diego, CA 92121
Phone: 858 450-7079 ...............................................
Fax: 858 550-1774
Web: http://www.cyntellect.com
CyTecs GmbH .............................................. 422, 424
Am Flugplatz 13
Gorlitz D-02828
Germany
Phone: 49 3581-8746 0
Fax: 49 3581 8746 70
Web: http://www.cytecs.com
CyTecs is a manufacturer of FCM modules and components (fluidics, mechanics, electronics and optics). Besides
this, anew dedicated class of ultracompact and mobile
FCM instruments was introduced in joint cooperation with
Partec for serving the rapidly growing demand of developing countries for a most stabile, robust and easy-to-operate as well as accurate and affordable alternative for
AIDS patient monitoring, offered at cost of only USD 2
per CD4 count.
81
Cytek Development, Inc. ........................... 303, 305
46560 Fremont Boulevard, Unit 115
Fremont, CA 94538
Phone: 510 657-0102
Fax: 510 657-0151
Web: http://www.cytekdev.com
De Novo Software ................................................. 210
64 McClintock Crescent Thornhill,
Ontario L4J 2T1, Canada
Phone: 905 738 9442
Fax: 310 943 1489
Web: http://www.denovosoftware.com
Cytek Development has been a leader in flow cytometry
upgrade products since 1989.
De Novo Software specializes in producing flow cytometry
data analysis software. Our flagship product, FCS Express, is a fully featured analysis package used by over
three hundred customers worldwide. FCS Express has
many powerful features including: creating Powerpoint
slides directly from your layout, post-acquisition compensation, unlimited undos and much more.
Upgrades include:
Additional color parameters
Automated Micro Sampling
FlowJo acquisition
20 liter Fluid Manager
Aerosol Containment
Calcium Flux Determination
Refurbished Cytometer Sales
Sample Line Filters
Sample Temperature Control
Superior Service/Repairs
Custom Instrumention
Duke Scientific Corporation ............................... 421
2463 Faber Place
Palo Alto, CA 94303
Phone: 650 424-1177
Fax: 650 424-1158
Web: http://www.dukesci.com
CYTOPEIA, INC. ........................................ 520, 522
12730 28th Avenue NE
Seattle, WA 98125
Phone: 206 364-3400
Fax: 206 364-3460
Web: http://www.cytopeia.com
Duke Scientific Corp offers powerful calibration and diagnostic tools for flow cytometry. Our Cyto-Cal calibration beads provide an easy and convenient method for
daily instrument setup and calibration. Our Cyto-Plex suspension array beads are available in three sizes in multiple
fluorescence levels and can accommodate up to 32 parameter analyses.
Cytopeia builds cell sorters for special research applications. inFlux®, our modular, high-performance platform
serves as a starting point for instruments of varying complexity from single-laser, routine analyzers to fully equipped
5-laser, multi-detector, high-speed sorters. Cytopeia offers a range of peripherals including a bio-containment
hood, special detectors and automated sampling units.
Evotec Technologies ............................................ 408
Hamburg D-22525, Germany
Phone: 49 40 560 81 0
Fax: 49 40 560 81 488
Web: http://www.evotec-technologies.com
Dako ........................................................................ 800
6392 Via Real
Phone: 800 235-5743
Fax: 805 566-6688
Web: http://www.dako.com
Fluid Imaging Technologies, Inc. ....................... 423
258 Cross Point Road
Edgecomb, ME 04556
Phone: 207 882-1100
Fax: 207 882-4800
Web: http://www.fluidimaging.com
Dako is a leading manufacturer of research and diagnostic solutions in the field of cancer. With a commitment to
the life sciences and the goal of optimizing patient care,
the company offers a diverse product line including reagents and instrumentation for anatomic and molecular
pathology, pharmacoDiagnostics, and flow cytometry.
Fluid Imaging Technologies manufacturers the
FlowCAM®, a continuous imaging flow cytometer for
analysis and photographic imaging of microscopic particles
contained in a fluid medium. FlowCAM data and highresolution images can be viewed real-time or retrieved
via patented VisualSpreadsheet© scattergram software for
robust and efficient analysis.
82
Fraen Corporation Srl .......................................... 310
Via E. Fermi,
7 Cusago 20090, Italy
Phone: 0039 02 90394049
Fax: 0039 02 90393736
Web: http://www.fraensrl.com
Fraen Corporation Srl as a supplier of advanced optical
solitions for lightning industry, develops fluorescence illuminators for commercial available microscopes based on
high power LEDs as fluorescence excitation source.
Fujitsu Computer Systems .................................. 306
200 Lowder Brook Drive, Suite 2100
Westwood, MA 02090
Phone: 781 326-6330
Fax: 781 326-8757
Web: http://us.fujitsu.com/biosciences
GCAT, Inc. .................................................... 325, 420
1629 Blue Spruce Drive, Suite 106
Ft. Collins, CO 80524
Phone: 800 720-GCAT
Fax: 970 472-9956
Web: http://www.gcatinc.com
GCAT is the exclusive distributor of Partec flow
cytometers/cell counters, reagents and consumables for
North America, providing sales, service and technical support. Partec’s low-cost, high-quality flow cytometers range
from single laser, single parameter instruments up to 4
lasers with 16 parameters and every Partec cytometer
performs absolute volumetric cell counting.
GE Healthcare ....................................................... 425
800 Centennial Avenue
P.O. Box 1327
Piscataway, NJ 08855-1327
Phone: 800 526-3593
Fax: 877 295-8102
Web: http://gehealthcare.com
Guava Technologies, Inc. ........................... 506, 508
25801 Industrial Boulevard
Hayward, CA 94545
Phone: 510 576-1400
Fax: 510 576-1500
Web: http://www.guavatechnologies.com
iCyt Mission Technology .................. 101, 103, 105,
............................................................... 200, 202, 204
P.O. Box 1593
Champaign, IL 61824-1593
Phone: 217 328-9396
Fax: 217 328-9692
Web: http://www.i-cyt.com
iCyt Mission Technology, maker of the Lyt laser system,
introduces revolutionary cell sorting technology with the
following features:
• Scalable systems with N-parallel sorting modules
• Sort rates exceeding 2 x 10^9 cells per hour
• Shared lasers and detectors across modules
• High speed digital signal processing
• Bio-safe operation (Class II, Type A2 cabinet)
Immunicon Corporation ....................................... 409
3401 Masons Mills Road, Suite 100
Huntingdon Valley, PA 19006
Phone: 215 830-0777
Fax: 215 830-0482
Web: www.immunicon.com
Immunicon Corporation is developing and commercializing proprietary cell-and molecular-based human diagnostic and life science research products with an initial focus
on cancer disease management. Immunicon has developed platform technologies for selection and analysis of
rare cells in blood, such as circulating tumor cells and circulating endothelial cells that are important in many diseases and biological processes. Immunicon’s products
and underlying technology platforms also have application
in the clinical development of cancer drugs and in cancer
research and may have applications in other fields of medicine, such as cardiovascular and infectious diseases.
Innovative Cell Technologies, Inc. .................... 214
6790 Top Gun Street, #1
San Diego, CA 92121
Phone: 858 587-1716
Fax: 858 453-2117
Web: http://www.innovativecelltech.com
Innovative Cell Technologies is celebrating 10 years in
business in 2006. ICT manufactures and sells cellular
declumping solutions for cell sorting, replacements for
trypsin that do not require washing or neutralization, primary tissue dissociation solutions and intracellular fixation
solutions for clumpy cells.
83
Invitrogen ..................................................... 415, 417
1600 Faraday Avenue
Carlsbad, CA 92008
Phone: 800 955-6288
Fax: 760-603 7229
Web: http://www.invitrogen.com
IQ Products ........................................................... 308
Rozenburglaan 13A
Groningen 9727 DL
The Netherlands
Phone: 31 50 5757 000
Fax: 31 50 5757 002
Web: http://www.iqproducts.nl
IQ Products develops and offers innovative diagnostic
applications in the fields of transplantation, perinatal diagnostics and routine flow cytometry.
ISAC Booth ........................................................... 311
History of the Society, Special Fulwyler Exhibit and Committee tables. The ISAC Booth is a special place for members to congregate and communicate.
Jackson ImmunoResearch Laboratories .......... 514
872 West Baltimore Pike, PO Box 9
West Grove, PA 19390
Phone: 610 869-4067
Fax: 610 869-0171
Web: http://www.jacksonimmuno.com
Affinity-purified secondary antibodies (many adsorbed
against other species for multiple labeling), new anti-mouse
IgG subclass specific, new anti-rabbit IgG light chain specific for Western blotting, anti-digoxin, anti-biotin, antiFITC, streptavidin, and purified immunoglobulins are conjugated with fluorophores (including Cy2, Cy3, and Cy5),
enzymes, Biotin-SP (long spacer), colloidal gold, phycoerythrin, and allophycocyanin.
JDSU ....................................................................... 314
430 North McCarthy Boulevard
Milpitas, CA 95035
Phone: 408 546-5000
Fax: 408 546-4300
Web: http://www.jdsu.com
JDSU designs, manufactures, and distributes commercial
laser components and subsystems for a broad range of
OEM applications in biotechnology instrumentation, graph84
ics and imaging, semiconductor water inspection, materials processing, remote sensing, and biohazard detection.
Our 355nm Xcyte is the world’s most precise and reliable
Quasi-CW solid state laser for flow cytometry applications < 200mW.
LightForm, Inc. ..................................................... 205
601 Route 206, Suite 26-479
Hillsborough, NJ 08844
Phone: 908 281-9098
Fax: 908 904-1067
Web: http://www.lightforminc.com
MAIA SCIENTIFIC ............................................. 320
Cipalstraat 3
Geel 2440
Belgium
Phone: 32 14 570620
Fax: 32 14 570621
Web: http://www.maia-scientific.com
MAIA SCIENTIFIC manufactures and markets novel
instrumentation and applications for high-throughput/high
information content screening. The MIAS®-2 microscopy
readers provide both brightfield and fluorescence parameters. Together with the eaZYX® imaging software the
system offers unprecedented sensitivity, speed and flexibility across the drug discovery and development value
chain.
Miltenyi Biotec ............................................ 516, 518
12740 Earhart Avenue
Auburn, CA 95602
Phone: 800 367-6227
Fax: 530 887-5399
Web: http://www.miltenyibiotec.com
Miltenyi Biotec is a diversified biotechnology company
offering comprehensive systems for biomedical research.
MACS® Technology has become the standard in magnetic cell separation worldwide, while MACS® Cell Analysis offers an extensive range of fluorochrome-conjugated
antibodies for flow cytometry. The MACSQuant™ Analyzer highlights Miltenyi Biotec’s commitment to innovative developments in flow cytometry instrumentation.
Molecular Devices ............................................... 218
1311 Orleans Drive
Sunnyvale, CA 94089
Phone: 800 635-5577 Fax: 408 747-3601
Web: http://www.moleculardevices.com
Molecular Devices Corporation is a leading supplier of
high-performance bioanalytical measurement systems that
accelerate and improve drug discovery and other life sciences research. Molecular Devices’ product solutions are
based on the company’s advanced core technologies that
integrate its expertise in engineering, molecular and cell
biology, electrophysiology, and chemistry.
MSP Corp…………………………………….402
5910 Rice Creek Parkway, Suite 300
Shoreview, MN 55126
Phone: 651 287-8100
Fax: 651 287-8140
Web: www.mspcorp.com
MSP Corporation is a global, applied particle technology
company providing advanced instruments, products and
services to the semiconductor, pharmaceutical, air pollution and biotechnology industries. We are known around
the world for offering ingenious solutions to complex problems involving the generation, deposition, sampling and/or
measurement of particles from 3nm to 100µm.
New Mexico Molecular Libraries
Screening Center .................................................. 403
UNM HSC CRF217, 2325 Camino De Salud
Albuquerque, NM 87131
Phone: 505 272-6206
Fax: 505 272-6995
Web: www.nmmlsc.health.unm.edu
The New Mexico Molecular Libraries Screening Center
(NMMLSC) is one of ten founding members of the NIH
Molecular Libraries Screening Center Network (MLSCN).
It is the only center specializing in high throughput flow
cytometry. The booth will provide a demonstration of
HyperCyt high throughput flow cytometry technology and
educational materials concerning the MLSCN and the NIH
Roadmap initiative.
Newport Corporation ........................................... 404
1791 Deere Avenue
Irvine, CA 92841
Phone: 949 863-3144
Fax: 949 253-1680
Web: http://www.newport.com
Newport is a premier global resource for customers that
need to make, manage, and measure light. Its SpectraPhysics division is a global leader and innovator in abroad
spectrum of lasers used in various bio-analytical
applications.We have unique, integrated expertise and
knowledge in both laser and white light sources, in optics
and optical mechanical positioning and detection and instrumentation.
Nikon Instruments Inc. ....................................... 211
1300 Walt Whitman Road
Melville, NY 11747
Phone: 631 547-8500
Fax: 631 547-8652
Web: http://www.nikonusa.com
Nikon will exhibit Live Cell Microscopy and Spectral
Imaging Instruments; Featured are the TE2000E2 with
Perfect Focus System enabling continuous focus eliminating thermal and mechanical focus drift during long-term
time lapse microscopy. C1si Spectral Imaging Laser Confocal System featuring fast, high resolution spectral
unmixing of multiple fluorescence probes. New Digital
Sight cameras and NIS-Elements imaging software for
microscopy.
Omega Optical, Inc. ............................................. 300
Delta Campus, Omega Drive
Brattleboro, VT 05301
Phone: 802 254-2690
Fax: 802 254-3937
Web: http://www.omegafilters.com
Omega Optical offers flow cytometry filters to support a
wide variety of fluorophores, tandems, and multi-color cell
sorting and analysis applications. Our flow cytometry filters are manufactured to fit all research and clinical instruments including Beckman Coulter, Becton Dickinson,
DAKO Cytomation, Partec, and others. We can manufacture custom filters to your specifications.
85
Parker Hannifin - Pneutronics Division ............ 203
26 Clinton Drive, Unit 103
Hollis, NH 03049
Phone: 603 595-1500
Fax: 603 595-8080
Web: http://www.parker.com/pneutronics
Picarro .................................................................... 201
480 Oakmead Parkway
Sunnyvale, CA 94085
Phone: 408 962-3900
Fax: 408 962-3200
Web: http://www.picarro.com
Partec GmbH ................................................ 321, 323
Otto-Hahn-Str. 32
Muenster, NRW D-48161
Germany
Phone: 49 2534 8008 0
Fax: 49 2534 8008 90
Web: http://www.partec.com
Picarro makes high performance, highly reliable lasers for
bio-instruments. The Picarro Cyan Laser is a compact,
solid-state 488 nm laser with typical MTTF > 45,000 hours
and output powers from 10 to 50 mW. Other laser wavelengths offered by Picarro include 375 nm, 405 nm and
632nm.
Partec, a worldwide leading flow cytometry instrumentation manufacturer and application solution provider in research and clinical routine, is a pioneer in Cell Analysis
having introduced the worldwide first commercial flow
cytometer (ICP-11) in 1968/69. Since then, the broad range
of Partec FCM systems from basic to high end configurations with up to 16 optical parameters and 5 light sources
is respected to offer the optimum of precision and accuracy.
Phoenix Flow Systems, Inc. ................................ 212
6790 Top Gun Street, Suite 1
San Diego, CA 92121-4121
Phone: 858 453-5095
Fax: 858 453-2117
Web: http://www.phnxflow.com
Phoenix Flow Systems manufactures and sells
Apoptosis(TUNEL, Annexin V) and Cell Proliferation
(anti-BrdU) reagent kits for flow & image cytometry, tissue dissociation and cell detachment products that replace
trypsin, CD-34, TdT & HLAB27 control cell products and
MultiCycle AV, state-of-the-art DNA ploidy and S-phase
fraction analysis software. Apo-BrdU, Apo-Direct,
VAnnexinV, Absolute-S, EZ-BrdU, Accutase, Accumax,
and CRISP are the names of these products.
86
ProZyme, Inc. ........................................................ 219
1933 Davis Street, Suite 207
San Leandro, CA 94577-1258
Phone: 510 638-6900
Fax: 510 638-6919
Web: http://www.prozyme.com
From the producers of phycobiliproteins and conjugates,
learn how to use our simple, flexible PhycoLink® Conjugation and Purification Kits to make the brightest, most
sensitive conjugates (R-PE, PerCP, APC and more). Join
us for a spirited discussion of factors critical for good
conjugates Tues, May 23rd at 13:00 in room 205B/C.
QImaging ............................................................... 109
4190 Still Creek Drive Burnaby,
BC V5C 6C6, Canada
Phone: 604 708-5061
Fax: 604 708-5081
Web: http://www.qimaging.com
QImaging is a world’s leading provider of high performance CCD digital FireWireTM cameras for use in life
science, OEM and industrial applications. Innovation and
reliability provide superior imaging for demanding applications in quantitative and high-resolution publication imaging. Continuing the tradition, come see our recently
released Retiga-SRV with iGlo Technology!
ScienceXperts, Inc. .............................................. 528
40479 Encyclopedia Circle
Fremont, CA 94538
Phone: 877 799-8811
Fax: 877 799-8811
Web: http://www.ScienceXperts.com
Spherotech Inc. ..................................................... 622
1840 Industrial Drive, Suite 270
Libertyville, IL 60048-9817
Phone: 847 680-8922
Fax: 847 680-8927
Web: http://www.spherotech.com
Email: [email protected] #:
ScienceXperts’ knowledge-based solutions help scientists
design and carry out laboratory studies. Its flagship product FacsXpert automates execution and management of
even the most complex FACS studies. Add the secure
DataStore and the Rescentris CERF electronic lab notebook for a complete solution to experiment design, data
collection, and collaborative data management.
Spherotech manufactures and supplies uniform, high quality
latex, fluorescent, paramagnetic, ferromagnetic and various colored microparticles for biomedical and diagnostic
applications. These microparticles are used in applications such as latex agglutination, fluorescence immunoassay, enzyme immunoassay, fluorescence microscopy, confocal fluorescence microscopy, flow cytometry, image
cytometry, magnetic cell separation, and magnetic particles.
Soft Flow Hungary Ltd. ........................................ 406
20 Kedves Street
Pecs, H-7628
Hungary
Phone: 36 72 240064
Fax: 36 72 240065
Web: http://www.softflow.com
Soft Flow develops and distributes PC and Mac versions
of FCAP Array, a scientific software for cytometric bead
array (CBA) technology. The software supports the flexible, open-architecture design of most commercially available microbead based technologies. FCAP Array can be
used with virtually any flow cytometer on the market.
SouthernBiotech ................................................... 419
P.O. Box 26221
Birmingham, AL 35260
Phone: 205 945-1774
Fax: 205 945-8768
Web: http://www.southernbiotech.com
Spectral Applied Research .................................. 504
10 North Rivermede Road
Concord, ON L4K 2H2, Canada
Phone: 905 326-5040
Fax: 905 326-5041
Web: http://SpectralAppliedResearch.com
Spectral Applied Research manufactures innovative laser illumination systems for microscopy and life science research.
The LMM5 laser merge module is a fiber-coupled source
containing up to five solid state lasers. Both shutter and AOTF
versions are available. Our optical expertise helps researchers solve problems that can lead to superior results.
Tree Star, Inc. ........................................................ 301
340 A Street, Suite 206
Ashland, OR 97520
Phone: 541 201-0022
Fax: 541 482-3153
Web: http://www.flowjo.com
FlowJo is a scientific analysis program designed for flow
cytometric data. It performs comprehensive analyses of
up to thousands of samples containing millions of events.
A wide array of visualizations and gating tools, special
platforms for kinetics, cell cycle analysis, quantitation, and
compensation is included. FlowJo produces the best publication quality charting available, and introduces new technologies for presenting and publishing flow data via the
web.
TTP LabTech Ltd. ................................................ 318
Melbourn Science Park, Cambridge Road
Melbourn, Royston
Herts SG8 6EE
U.K.
Phone: 44 1763 262626
Fax: 44 1763 261964
Web: http://www.ttplabtech.com
TTP LabTech’s Acumen Explorer fluorescence microplate
cytometer offers fast high content screening for protein
kinase activation, cell cycle analysis and reporter gene
expression. It scans the whole well for 100% data confidence, collecting data for up to 4 colours simultaneously.
Typical scan and analysis times are under 10 minutes a
plate.
87
Union Biometrica, Inc. ......................................... 304
84 October Hill Road
Holliston, MA 01746
Phone: 508 893-3115
Fax: 508 893-8044
Web: http://www.unionbio.com
Wiley ....................................................................... 400
111 River Street
Hoboken, NJ 07030
Phone: 201 748-6000
Fax: 201 748-6088
Web: http://www.wiley.com
Union Biometrica manufactures COPAS BioSorter flow
cytometers for analysis, sorting, and dispensing of “large”
objects (approximately 20–1500µm) such as large cells,
cell clusters, beads, seeds, C.elegans, Drosophila, etc. The
large-bore flow cell and non-destructive pneumatic sorting mechanism permit sorting of objects traditionally considered too large/fragile for conventional flow cytometry.
Founded in 1807, John Wiley & Sons, Inc., provides musthave content and services to customers worldwide. Its
core businesses include scientific, technical, and medical
journals, encyclopedias, books, and online products and
services; professional and consumer books and subscription services; and educational materials for undergraduate
and graduate students and lifelong learners.
Verity Sofware House .................................. 427, 429
45A Augusta Road, P.O. Box 247
Topsham, ME 04086
Phone: 207 729-6767
Fax: 207 729-5443
Web: http://www.vsh.com
Verity Software House develops flow cytometry analytical software (PC and Macintosh) for comprehensive
immunophenotyping, DNA cell cycle analysis, and quantitative cytometry. WinList™, ModFit LT™, and
QuantiCALC™ are ready to go to work for you today.
Come visit us in booths 427-429 for a look at exciting new
developments in REMOTE CYTOMETRY!
DISLAIMER:
Participation in the Exhibits Program does not constitute an endorsement by the International Society for Analytical Cytology (ISAC) of the claims, products or services offered.
88
Abstracts
Keynote Session
1
MOLECULES CRAFTED TO SPY ON CELLS AND
TUMORS
Roger Y. Tsien1
1
University of California, San Diego, Pharmacology, School of
Medicine, La Jolla, California
Cell Profiling of Potentiated Phospho-Protein Networks in Cancer Cells.
Cell. 118:217-228. (2) Sachs K., Perez O., Pe’er D., Lauffenburger
D.A and Nolan G.P. 2005. Causal protein-signaling networks derived
from multiparameter single-cell data. Science. 308:523-9.
3
LOCATION PROTEOMICS: IMAGE INFORMATICS FOR
SYSTEMS BIOLOGY
Robert F. Murphy1
1
Genetically encoded tags and indicators are molecular spies that reveal
specific gene products and biochemical processes in living cells and
organisms. Fluorescent proteins from jellyfish and corals have been
bred to eliminate multimerization and cover the entire visible spectrum.
Somatic hypermutation in B lymphoma cells harnesses the immune
system to produce a powerful new way to evolve protein properties.
Indicators constructed from fluorescent proteins can report local dynamic
signals such as redox potential, neurotransmitter concentrations, proteinprotein interactions, and kinase vs. phosphatase activities.
Although
fluorescent proteins are powerful tools, they cannot be reduced below
~220 amino acids, and their only useful readout is fluorescence. Much
shorter peptide sequences combine genetic encoding with the greater
range of spectroscopic properties available by organic synthesis.
Tetracysteine motifs of 6-12 amino acids can be labeled in live cells
with membrane-permeant biarsenical dyes. Unique applications include
green vs. red pulse-chase labeling of old vs. new copies of the same
protein, electron-microscopic localization, chromophore-assisted light
inactivation of a chosen protein without the problems of antibody
penetration, and measurement of local Ca2+ within nanometers of proteins
such as Ca2+ channels.
For clinical applications one would prefer not
to have to introduce genes or be limited to optical detection. Argininerich sequences are known to mediate uptake of a wide variety of contrast
agents into cells and tissues in vivo. We find that such uptake can be
prevented by appending certain polyanionic sequences and selectively
re-activated by cleavage of the linker. This new mechanism offers the
exciting possibility that radioactive, magnetic, and infrared contrast
agents and therapeutic drugs may be concentrated in diseased tissues
expressing particular extracellular proteases.
2
SINGLE CELL KINASE SIGNALING FOR MECHANISTIC
AND CLINICAL ANALYSES
Garry P. Nolan1
1
Stanford University, Microbiology and Immunology, School of
Medicine, Stanford, California
Intracellular assays of signaling systems has been limited by an inability
to correlate functional subsets of cells in complex populations based on
active kinase states or other nodal signaling junctions. Such correlations
could be important to distinguish changes in signaling status that arise
in rare cell subsets during functional activation or in disease
manifestation. Simultaneous detection of activated kinases and
phosphoproteins in simultaneous pathways in subpopulations of complex
cell populations by multi-parameter flow cytometric analysis allows
identification of signaling cascades for disease states by ordering of
kinase activation and phosphoprotein status in signaling hierarchies.
Importantly, we demonstrate that ordering of these activations requires
multiple interrogations of cells, and that the networks discovered are
reflective of deeper correlations. Using Bayesian Network analysis (a
form of machine learning) one can infer pathway connectivity in an
automated fashion, allowing for high throughput derivations of signaling
system networks graphs in PRIMARY CELLS. Notably, when kinase
inhibitors, previously selected in in vitro assays are tested on complex
cell populations, single cell analysis of signaling states reveals shocking
differences in the inhibition of kinase activity in different cell subsets
that will be discussed. The approach has powerful applications in
mechanistic understanding, drug screening, and patient stratification
for prediction of disease outcome in cancer, autoimmunity, infection,
based on signaling network status. (1) Irish J.M., Hovland R., Krutzik
P.O., Perez O.D., Bruserud O., Gjertsen B.T., Nolan G.P. (2004) Single
Carnegie Mellon University, Biological Sciences & Biomedical
Engineering, Mellon College of Science & Carnegie Institute of
Technology, Pittsburgh, Pennsylvania
Systems Biology requires comprehensive, systematic data on all aspects
and levels of biological organization and function. In addition to
information on the sequence, structure, activities, and binding
interactions of all biological macromolecules, the creation of accurate,
predictive models of cell behavior will require detailed information on
the distributions of those molecules within cells and the ways in which
those distributions change over the cell cycle and in response to mutations
or external stimuli. Current information on subcellular location in
protein databases is limited to unstructured text descriptions or sets of
terms assigned by human curators. These entries do not permit basic
operations that are common to other biological databases, such as
measurement of the degree of similarity between the distributions of
two proteins, and they are not able to fully capture the complexity of
protein patterns that can be observed. The field of location proteomics
seeks to provide automated, objective, high-resolution descriptions of
protein location patterns within cells. The initial foundation for the
field was the demonstration that automated classifiers could be trained
to recognize all major subcellular patterns in fluorescence microscope
images, and especially the critical finding was that such systems could
discriminate location patterns better than visual examination. The very
high accuracy (over 98% on single 3D images) of these systems gave
confidence that the numerical features used to describe location patterns
could form a basis for extending the methods to unsupervised learning
of patterns. To this end, we have described grouping of proteins into
statistically-indistinguishable location patterns using consensus clustering
methods. The resulting clusters, or location families, are analogous to
clusters found for other domains, such as protein sequence families.
Our current work is focusing on extending this work in a number of
new directions. These include analyzing the temporal dependence of
subcellular patterns, generalizing pattern analysis across many cell types,
analyzing mixed multicell images in cultures and tissues, and creating
generative models of subcellular patterns that can be incorporated into
comprehensive models of cell behavior. The combination of these
methods with large scale, high throughput imaging approaches will
allow realistic cell modeling that reflects detailed knowledge of the
subcellular location of all proteins. We anticipate that work in this field
will also lead to improved diagnostics based on subcellular pattern
discrimination.
4
THE NIH CHEMICAL GENOMICS CENTER:
ANNOTATING THE BIOLOGICAL ACTIVITY OF
CHEMICAL LIBRARIES USING QUANTITATIVE HIGH
THROUGHPUT SCREENING
Jim Inglese1
1
National Institutes of Health (NIH), NIH Chemical Genomics
Center, Bethesda, Maryland
The NIH Chemical Genomics Center (NCGC; www.ncgc.nih.gov/) is
the founding member of the Molecular Libraries Screening Center
Network (MLSCN), a network of ten centers established as part of the
NIH Roadmap for Medical Research (nihroadmap.nih.gov/). The
mission of the NCGC is to make accessible the technologies and protocols
of high throughput biomolecular screening and chemistry optimization,
developed primarily in the pharmaceutical and biotech industries, to
academic investigators. The ‘product´ emerging from the NCGC pipeline
will not be drugs, but rather chemical probes to aid in the understanding
of biology and validation of therapeutic targets. In refining the screening
process, the NCGC has developed a strategy called “quantitative HTS”
89
(qHTS) to generate concentration-response curves for a range of
biochemical and cellular assays on large compound collections using
existing technologies. Our process is highly refractory to false positives,
easily identifies compounds of low potency and efficacy, and
demonstrates the potential to build high-quality chemical genomic
databases. Examples from this new paradigm will be presented.
5
HIGH THROUGHPUT FLOW CYTOMETRY AND THE NIH
ROADMAP MOLECULAR LIBRARIES INITIATIVE
Larry A. Sklar1, Jeffrey Arterburn2, Bruce Edwards3, Tudor I
Oprea4, Eric R Prossnitz5, Herbert G Tanner6
1
University of New Mexico, Cancer Researcn and Treatment Center
and Pathology, Health Sciences Center, Albuquerque, New Mexico;
2
New Mexico State University, Chemistry and Biochemistry, Las
Cruces, New Mexico; 3University of New Mexico, Cancer Research
and Treatment Center and Pathology, Albuquerque, New Mexico;
4
University of New Mexico, Biochemistry and Molecular Biology,
Albuquerque, New Mexico; 5University of New Mexico, Cell
Biology and Physiology, Albuquerque, New Mexico; 6University of
New Mexico, Mechanical Engineering, Albuquerque, New Mexico
The high throughput (HT) flow cytometry platform HyperCyt is adept
at both cell and particle-based assays and is compatible with both high
content and multiplex analysis. Our cell-based assays have initially been
directed against G protein-coupled receptor (GPCR) targets where we
have identified novel small molecule ligands for peptide and steroid
receptors. We have developed general particle-based multiplexed
approaches compatible with assemblies of soluble membrane receptors,
receptor tails, proteases, kinases, nucleases, etc. We have probed the
mechanism of partial agonism and the steps in signal transduction using
flow cytometry-based kinetic analysis with soluble GPCR. We have also
developed approaches probing cell-cell adhesion, nanoscale integrin
conformational changes, and responses to nanoscale intercellular forces.
The NIH Roadmap Molecular Libraries Initiative (MLI) has given us
the opportunity, through the New Mexico Molecular Libraries Screening
Center (NMMLSCN, http://nmmlsc/) to implement HT flow cytometry
for the international research community. The MLSCN has expertise in:
1) target and assay development; 2) the integration of flow cytometric
and virtual screening to enhance the discovery process; and 3) medicinal
chemistry for the optimization of active molecules and the development
of imaging agents. Through MLI and collaborations with investigators
outside the MLSCN, we are currently developing cell-based assays for
cytotoxicity where both cell viability and cell number can be recorded,
bacterial virulence, multi-drug resistance, androgen response, protein
expression, and generic cell responses, to name a few. We are also
working on particle-based multiplexed protein-protein assays for
interactions between Bcl-2 family members and generic proteinoligonucleotide interactions.
6
THE PHARMACODYNAMICS OF MOLECULAR CANCER
THERAPEUTICS
David Hedley1
1
Princess Margaret Hospital, Toronto, Ontario, Canada
Our approach to cancer is being revolutionized through rapid progress
understanding the molecular mechanisms, coupled to rational drug design
programs producing highly selective agents to target these mechanisms.
Despite the current excitement, there are formidable obstacles to making
effective molecular cancer therapeutics a clinical reality. Unlike classical
chemotherapy and radiotherapy, the drugs are highly selective in their
actions, and effective only when their molecular target is playing a
significant role driving the malignant process. Due to the complex,
multigenetic basis of cancer development, it is unlikely that a single
molecular targeted agent could achieve long term cancer control in the
clinic. More likely, optimal treatment will consist of combinations of
agents, rationally selected based on understanding the downstream
interactions of drug targets, and individualized by analysis of the patient´s
tumour tissue. Fluorescence-based techniques using flow or image
cytometry have major advantages studying complex biology, because
they address the problems of cellular heterogeneity, and are able to
study molecular interactions through the use of multiple fluorescence
labels. The development of phosphospecific antibodies has allowed the
introduction of techniques to study signal transduction at the single cell
level, including the analysis of complex signaling interactions. This is
90
of particular importance to molecular oncology, since a large number
of agents currently in clinical trial inhibit signaling pathways. As well as
measuring baseline activity to identify if the drug target is being expressed
in the cancer cells, cytometric methods can also be used to monitor
pharmacodynamic effects during treatment. Pharmacodynamics is a
branch of pharmacology that asks how drugs impact on the host tissues,
whereas pharmacokinetics studies how drugs are metabolized and
eliminated by the patient. During the early phases of drug development,
it is important to show that the drug is interacting with its target in vivo,
and to determine the relationships between drug dose and the extent of
target inhibition. Along with others, our group has been able to develop
pharmacodynamic markers based on flow cytometry and fluorescence
image analysis, validate these using experimental systems, and then
translate the methods to the actual clinical situation. Pharmacodynamic
information is critical to understanding what is going on in a patient´s
cancer cells during treatment, in order to explain why treatment works
in some patients but not in others. Although still in its infancy, cytometrybased pharmacodynamics has the potential to play a major role bridging
between basic science, pathology, pharmacology, and molecular
oncology.
7
SEARCHING FOR AN HIV VACCINE: THE ROLE OF
FLOW CYTOMETRY
Mario Roederer1
1
ImmunoTechnology Section, Laboratory of Immunology, Vaccine
Research Center, NIAID, NIH, Bethesda, Maryland
Vaccine development to protect against HIV development is actively
proceeding on two fronts: generation of a sterilizing (neutralizing)
humoral response, and generation of an effective cellular response. To
date, it has been impossible to generate an antibody response of sufficient
potency that sterilizing vaccination is possible. Nonetheless, a vaccine
can be considered successful on a global scale if the induced cellular
response is sufficient to dampen viral loads (reducing transmission) as
well as reducing morbidity and mortality after infection. We use a
variety of adjuvants, delivery mechanisms, and immunogens to induce
a variety of T cell responses. By challenging vaccinated nonhuman
primates with live virus, we hope to identify the kinds of responses that
are best correlated with protection as measured by a reduction in peak
viral load, set point viral load, and amelioration of the dramatic destruction
of memory CD4 T cells during acute infection. As we move forward
through phase I and II clinical trials in humans, and prepare for phase
III, we are keen to determine whether or not these types of responses are
induced in humans as well. The primary tool for determining the
quantity and quality of the T cell response is flow cytometry. Using
sophisticated instrumentation, software, reagents, and techniques, we
are able to measure five or more different functional responses
simultaneously from each cell (e.g., cytokine profile, cytotoxic potential,
proliferative capacity). These complex combinations of functions reveal
that there are a number of distinct “flavors” of T cell responses present
in either naturally infected or vaccinated subjects; the task now is to
identify which of these flavors is most suited to protection from
challenge. In addition, by studying rapid HIV progressors vs. long
term nonprogressors, we are beginning to identify differences in T cell
responses that are correlated with disease pathogenesis, perhaps pointing
us towards desirable types of vaccine responses. Significant challenges
remain, however. The automation of the sample analyses, the automation
of the data analyses, compliance with GLP, and validation of these
procedures is an enormous challenge. Integration and presentation of
the highly complex datasets is still intensely laborious and challenging.
In these terms, flow cytometry technology development is shifting from
the instrumentation to the software and data interpretation aspects, which
have yet to catch up to the rapid growth of the hardware capabilities.
8
FRONTIER LECTURE 4: HUMAN STEM CELL BIOLOGY
AND POTENTIAL APPLICATIONS
Mickie Bhatia1
1
McMaster University, Hamilton, Ontario, Canada
Dr. Bhatia’s program has characterized signaling pathways that regulate
human stem cells and initiated new approaches of changing human
stem cell behaviour. Using human to mouse xenotransplant systems to
define human stem cells in the whole body, he has shown that growth
factors normally abundant during fetal development can be introduced
to adult stem cells and change their growth properties and ability to
form other cell types. In parallel, Dr. Bhatia has demonstrated that cord
blood stem cells have the potential of regenerating a damaged pancreas
and correcting diabetic-like symptoms. Where human embryonic stem
cells (hESCs) have been explored, his work provides evidence of the
ability to generate blood stem cells from hESC lines and novel ways of
growing hESC lines in culture. These findings provide novel procedures
that may be useful in cell transplantation based regenerative therapies
and continue to compare the effectiveness of adult and embryonic stem
cells alike.
9
THE LCOS-BASED PROGRAMMABLE ARRAY
MICROSCOPE (PAM)
Thomas M. Jovin1, Martin Thomas2, Guy M. Hagen1, Donna
J. Arndt-Jovin3, Keith A. Lidke4, Andrew Hill2, A. Martyn
Reynolds2, Jeremy Graham2, Rainer Heintzmann5, Quentin
Hanley6
1
Max Planck Institute for Biophysical Chemistry, Molecular
Biology, Goettingen, Niedersachsen, Germany; 2Cairn Research
Ltd., Faversham, United Kingdom; 3Max Planck Institute for
Biophysical Chemistry, Goettingen, Niedersachsen, Germany;
4
Sandia National Laboratories, Biomolecular Materials and
Interfaces Department, Albuquerque, New Mexico; 5King’s College
London, New Hunt’s House, Randall Division of Cell and
Molecular Biophysics, London, United Kingdom; 6Nottingham
Trent University, School of Biomedical and Natural Sciences,
Nottingham, United Kingdom
In 1998, we reported the realization of an optically sectioning
fluorescence microscope (PAM) for rapid, light efficient 3D imaging of
living specimens. 1,2 The design was based on conjugate structured
illumination & detection implemented with a spatial light modulator
(SLM) located at an image plane of a conventional fluorescence
microscope. The major advantages of the PAM are: (1) compact design
with no moving parts; (2) very significant speedup/improvement in
sectioning due to a pixel duty cycle of up to 50%, and simultaneous
detection & processing of both conjugate (in-focus) & non-conjugate
(out-of-focus) images; (3) ultrasensitive detection via electron
multiplying CCD cameras; (4) programmable & adaptive optical
sectioning based on libraries of dot, line & pseudo-random (Sylvester)
sequence patterns; (5) flexible light sources, including LEDs; (6)
generation & detection of arbitrarily polarization patterns; (7)
compatibility with hyperspectral & lifetime-resolved imaging; (8)
incorporation of light sources for photo-destruction and/or
transformation, modes compatible with FRAP, FLIP & FCS/ICS; (9)
minimal photobleaching. We report here the design, operation &
application(s) of a new generation commercial PAM created by the
combined efforts of the MPIbpc (Mol Biol Dept) and Cairn Research
Ltd. (UK). The stand-alone module, including light source(s) and
detector(s), features an innovative catadioptric design and a ferroelectric
liquid-crystal-on-silicon (LCoS) SLM instead of the original DMD. It
can be attached to a side port of a(ny) unmodified fluorescence
microscope. The Cairn prototype system currently operated at the MPI
incorporates a 6-position high-intensity LED illuminator and an Andor
iXon emCCD camera, and is mounted on an Olympus IX71 inverted
microscope with 60-150x objectives and a PZ translator, a Cairn
Optoscan monochromator, and a Prior Scientific ProScan stage. Further
enhancements are being incorporated: (i) point- and line-wise spectral
resolution, detecting with an Applied Scientific Imaging SpectraCube
imager or a Specim ImSpector imaging spectrograph; and (ii) lifetime
imaging (FLIM) using phase-modulation 2 and TCSPC modules.
Multiphoton operation and other nonlinear techniques should be feasible.
Real-time sectioning with 50-100 ms exposures and lag-free manual
focusing have been achieved with a single LED source. Operation is
insensitive to mechanical shock applied to the microscope table; thus,
the system is suitable for ruggedized (field) use. It is currently being
applied to developmental biology and signal transduction studies, e.g.
based on quantum dot ligands 3 . 1 Verveer PJ et al. 1998. J.
Microsc.189:192; 2Hanley QS et al. 2005. Cytometry 67A:112 (latest
PAM paper); 3Lidke DS et al. 2005. J. Cell Biol. 170:619.
10
CHEMICAL CYTOMETRY-ANALYTICAL CHEMISTRY
OF SINGLE CELLS
Norman Dovichi1
1
University of Washington, Chemistry Department, Seattle,
Washington
It is now possible to generate one- and two-dimensional protein
electrophoresis data from single mammalian cells. These separations
are capable of resolving a hundred components from the cell, and the
data can be correlated with cell cycle and other properties of the cells.
Examples have been generated from breast, prostate, colon, and
esophageal cancer cell lines, from macrophages, astrocytes and neurons,
and from osteoprecursors and myoblasts. Instrumentation is under
development that can characterize tens of thousands of cells per day in
one-dimensional electrophoresis and thousands of cells per day in twodimensional electrophoresis. Other instrumentation is able to detect
specific proteins at the single copy level in single cells and determine
post-translational modifications of that protein. Finally, metabollic
pathways for both carbohydrates and glycolipids have been monitored
in single cells.
Plenary Sessions
11
MULTIMODE LIVE CELL IMAGING REVEALS A NOVEL
METHOD OF CELLULAR COMMUNICATION IN THE
IMMUNE SYSTEM
Simon C. Watkins1
1
University of Pittsburgh, Center for Biologic Imaging, School of
Medicine, Pittsburgh, Pennsylvania
Engagement of cell surface receptors leads to intracellular signaling
that has generally been shown to alter phenotype and function of
individual cells. Using a variety of cutting edge optical imaging methods
We show here that myeloid-lineage dendritic cells and monocytes can
be triggered to flux calcium by mechanical contact with a microprobe,
and that the signal can be propagated within seconds to other cells at
distances up to a hundred microns away by membranous connections
called tunneling nanotubules (TNTs). These form a complex and
transient network in live cells, with individual TNTs exhibiting great
variation in length and diameter. In addition to calcium fluxes,
microinjected dye tracers can be transferred through these connections.
Following TNT-mediated stimulation, spreading of lamellipodia occurs
in dendritic cells characteristic of that seen during the phagocytic response
to bacteria. These results demonstrate that functional signals generated
in a single cell can be transmitted to other cells through a physically
connected network.
12
FLUORESCENT SPECKLE MICROSCOPY
Gaudenz Danuser1
1
The Scripps Research Institute, Department of Cell Biology, La
Jolla, California
Fluorescent speckle microscopy (FSM) is a method to analyze the
movement and assembly/disassembly dynamics of macromolecular
structures. It capitalizes on fluorescent analog cytochemistry, in which
purified protein is covalently linked to a fluorophore and microinjected
or expressed as a GFP fusion in cells. Fluorescent protein coassembles
with unlabeled, endogeneous protein, visualizing the localization and
architectural organization of the structure inside a living cell. Despite its
broad application, this classic approach has been limited in reporting
subcellular dynamics because of the inherently high background
fluorescence from unincorporated and out-of-focus incorporated
fluorescent subunits and the uniform labeling of the target structure.
However, if the labeled subunits make up less than 1% of total pool of
subunits, fluorophore patterns of high non-uniformity accumulate. They
provide a unique random code identifying specific subareas of the
target structure in space and time. Addition of new fluorophores to the
code indicates the local association of subunits; subtraction of
fluorophores the local dissociation. These molecular events can be
monitored by diffraction-limited imaging using wide-field, total internal
reflection, or spinning disc confocal fluorescence microscopy equipped
with sensitive CCD cameras. The resulting images display punctate
patterns of local maxima, called speckles, over a dimmer background.
91
Speckles correspond to regions of the size of the point-spread-function
with a higher fluorophore density and serve as fiduciary marks of the
dynamics of the underlying structure. Although conceptually simple,
the interpretation of FSM time-lapse sequences requires sophisticated
computational tools for the tracking and statistical analysis of the
intrinsically stochastic and highly convoluted signals. In this lecture I
will cover the basic design of algorithms that simultaneously track
thousands of speckles; and statistical models that allow the conversion
of positional fluctuations and speckle intensity variations into
spatiotemporally resolved maps of transport, turnover, and viscoelasticity
of the probed structure. I will then report how quantitative FSM yielded
major discoveries of the dynamic organization of the actin cytoskeleton
in migrating cells and of the transient interaction of the cytoskeleton
with other cellular assemblies such as focal adhesions. With a second set
of examples I will indicate how we exploit the submicron resolution of
this technique to infer molecular mechanisms of force generation during
cell migration. Finally, I will illustrate the power of FSM as a general
quantitative method for imaging the dynamics of any multiprotein
structure in cells and in vitro.
13
FACILE GENERATION OF BIOSENSORS TO STUDY
ENDOGENOUS PROTEIN ACTIVATION IN LIVING
CELLS
Klaus Hahn1
1
University of North Carolina at Chapel Hill, Department of
Pharmacology, Chapel Hill, North Carolina
We are developing new methods for studying the spatio-temporal
dynamics of signaling with minimal perturbation. By conjugating novel
dyes to affinity reagents, activities of endogenous proteins can be studied
with high sensitivity. This approach opens the door to high throughput
generation of biosensors via phage display and other screening for
biosensor affinity elements. The approach also readily permits analysis
of multiple signaling activities in the same cell.
14
MULTIPLEXED HYBRID CYTOMETRY BASED
APPLICATION PLATFORM TECHNOLOGY: A TOOL TO
FIGHT THE HIV PANDEMIC IN THE 21ST CENTURY
Francis Mandy1
1
Health Canada, Ottawa, Ontario, Canada
In sub-Saharan Africa about 12 million people perish every year from:
HIV, malaria and tuberculosis. For the majority of the individuals who
make up these gruesome annual statistics, the cause of death is
uninvestigated. The introduction of affordable antiretroviral therapy
(ART) in parts of Africa provides an opportunity to establish infrastructure
to support laboratory medicine. This movement is a significant step in
the direction of an effective health care system. Conventional laboratory
medicine from resource rich regions is incompatible with African
economical, cultural and political realities. In sub-Saharan countries
38% of the population exists on <$1 a day, with a gross national income
per capita <$500. In the past, in most resource limited regions, founding
agencies’ concentrated on disease prevention and provision of care.
Until now, little effort has been exerted to build sustainable laboratory
capacity. With the introduction of ART in Africa, health care policy
makers and clinical scientist recognize the urgent need for affordable
and sustainable laboratory infrastructure to support the diagnosis and
treatment of HIV. For 25 years, flow cytometry has been the CD4 T-cell
enumerating devic, to monitoring immune status of HIV infected
individuals. Recently, numerous companies introduced affordable and
robust cytometers both the flow and none-flow variety. Currently, the
lower cost, dedicated instruments are the most popular choice for CD4
T-cell counting in Africa. Accumulative sales of these instruments are
reaching significant numbers. When establishing affordable and
sustainable comprehensive laboratory infrastructure is the overall long
term objective; they in fact may turn out to be not the most cost effective
option. In this presentation features of the hybrid flow based application
platform (HyFAP) will be covered. The focus is on how to harness
HyFAP and make economical sense in sub-Sahara Africa. They run
both cell and microsphere based assays. Evidence will be presented to
illustrate how a multi-functional and multiplexing capacity is a realistic
option in resource poor countries. In the future, HyFAP will be connected
to GSM wireless external quality monitoring services (WEMS). The
integration with WEMS will assure minimum global standards for
immunology laboratories. With HyFAP, it is possible to build laboratory
92
capacity to provide rapid, accurate affordable and reliable diagnostic
tests to battle infectious diseases in most resource poor regions of the
globe.
15
REGULATION AND THERAPEUTIC TARGETING OF
HUMAN LEUKEMIA STEM CELLS
Kristin Hope1, Liqing Jin2, Eric Lechman2, John Edgar Dick3
1
University of Toronto, Toronto, Ontario, Canada; 2University
Health Network, Cell and Molecular Biology, Toronto, Ontario,
Canada; 3University of Toronto, Medical Genetics & Microbiology,
Faculty of Medicine, Toronto, Ontario, Canada
In acute myeloid leukemia (AML) the leukemic clone is organized as a
hierarchy originating from rare leukemic stem cells (LSC). Our interest
has therefore been in identifying and therapeutically exploiting the
molecular mechanisms that are uniquely required by these LSC.
Although gene expression analysis is a common way of identifying the
molecular players in stem cell function, few have explored the potential
for microRNAs (miRNAs) in such a regulatory role. MiRNAs are 22
nucleotide (nt) non-coding RNAs processed from hairpin precursors
that regulate translational repression of target genes. MiRNAs have
been implicated in directing diverse biological processes including
neoplasia. The identification of a set of embryonic stem cell specific
miRNAs suggests that these may be involved in maintenance of the
pluripotent state while a study linking miRNAs to fate determination
showed ectopic expression of a specific miRNAs in hematopoietic
progenitors could promote B or T cell differentiation. To address the
role of miRNAs in the regulation of LSC we performed miRNA array
analysis on 4 purified fractions based on CD34 and CD38 expression
from 6 primary AMLs. We identified a unique miRNA signature that
discriminates the CD34+CD38- fraction from more mature populations.
One candidate, mir155, was also found to be differentially highly
expressed in the stem cell fraction of normal cord blood by affymetrix
array. RNAi-mediated knockdown of mir155 in a novel CD34+ leukemic
cell line resulted in a loss of CD34 expression, an increase of
differentiation antigen expression and significantly reduced proliferative
potential. Our results are suggestive of a role for microRNAs in the
regulation of LSC and leukemogenesis.
In order to cure AML, LSC
must be effectively targeted, however as existing therapeutic strategies
target cycling cells and LSC are largely quiescent, new approaches must
be found. We have shown previously that an activating monoclonal
antibody (H90) directed against the adhesion molecule CD44 can release
the AML differentiation block when administered in vitro. To address
whether CD44 activation can act at the level of the LSC, H90 was
administered to NOD/SCID mice transplanted with primary AML cells.
We show that H90 greatly impairs leukemic repopulation by up to 95%
compared to mice injected with control antibody. In addition, posttreatment grafts exhibited a much reduced level of primitive cells and
an increase in immunophenotypically differentiated cells. The absence
of leukemia in serially transplanted mice established direct targeting of
LSC. The mechanism of H90 induced LSC loss involves both a promotion
of LSC commitment and an impairment of their homing to supportive
microenvironments.
16
DIAGNOSING PNH AND PREVIOUSLY UNDIAGNOSED
ACUTE LEUKEMIAS WITH FLAER AND
MULTIPARAMETER FLOW CYTOMETRY
D. Robert Sutherland1, Nancy Kuek1, Jeff Davidson1, Sylvia
Lynn Bamford2, Michael Keeney3
1
University Health Network, Clinical Flow Cytometry, Toronto,
Ontario, Canada; 2London Health Sciences Centre, Flow Cytometry,
London, Ontario, Canada; 3London Health Sciences, Hematology/
Flow Cytometry, London, Ontario, Canada
Paroxysmal Nocturnal Hemoglobinurea (PNH) is an acquired
Hematopoietic Stem Cell disorder caused by a somatic mutation in the
X-linked pig-a gene. This results in a partial or absolute deficiency of
all glycophosphatidyl-inositol (GPI)-linked proteins/glycoproteins. The
classical approach to diagnosis of PNH by cytometry involves the loss
of at least 2 GPI-linked antigens (typically CD55 and CD59) on two
different cell lineages (RBCs and Neutrophils). The bacterial lysin
Aerolysin binds to the GPI moiety of cell surface GPI-linked molecules
and causes lysis of normal cells but not GPI-deficient PNH cells. FLAER
is a fluorescently-labeled inactive variant of aerolysin that does not
cause lysis of cells to which it binds and this reagent is becoming more
widely used in the diagnosis of PNH by Flow. In a single tube assay, we
have combined FLAER with CD45, CD33 and CD14 that allows the
simultaneous analysis of FLAER and the GPI-linked CD14 structure on
neutrophil and monocyte lineages. Comparison to standard CD55 and
CD59 analysis shows excellent agreement. The assay can be performed
up to 48 hours after sample draw and data interpretation is
straightforward. Additionally, we were able to detect fewer than 5%
PNH-like monocytes and neutrophils in several cases of aplastic anemia,
which we were otherwise unable to detect using CD55 and CD59 on red
blood cells. Due to the higher signal to noise ratio, the method shows
increased sensitivity in our hands over single (CD55 or CD59) parameter
analysis. Interestingly, we have also detected leukemic blasts which
show aberrant FLAER staining in several samples sent to us for ‘PNH
testing’ including 1 case each of AML-M1, erythroleukemia (M6), a
case of acute leukemia developed in a patient with a history of CMML
and two cases of acute myelomonocytic leukemia. In all of these cases,
the neutrophils stained normally with FLAER, thereby ruling out PNH,
while the gated CD33bright cells failed to express CD14 and bound
lower levels of FLAER.
17
HIGH CONTENT ANALYSIS IN DRUG DISCOVERY
Joseph Trask1
1
Abbott Laboratories, Target and Lead Discovery, Abbott Park,
Illinois
This talk will describe past, current and future trends of the use of highcontent analysis (HCA) and high-content screening (HCS) in the
biopharmaceutical industry and the crossover of the technology into
the academic arena. The term “HCA/HCS” typically describes automated
fluorescent imaging, image analysis, data management and the
applications thereof. The technology not only complements flow
cytometry technology, it resembles flow cytometry during its infancy
with many challenges and a rapidly changing future. The impact of
HCA/HCS technology in the biopharmaceutical industry is being felt
throughout the entire drug development process from early drug
discovery, target identification, target validation, screening through
preclinical biomarker discovery including toxicology and mechanism
of action studies. The technology has cross-pollinated into many areas
of science including basic science and even material sciences. The goal
of the talk is to provide the audience with a brief history of HCA/HCS
and case studies where the technology has made an impact.
18
RARE EVENT DETECTION IN SOLID CANCERS
19
IMAGE-BASED SCREEN FOR CELL CYCLE AND
CANCER TARGETS
Daniel R. Rines1
1
Genomics Institute of the Novartis Research Foundation (GNF),
GNF Bio-Imaging Center, San Diego, California
Chromosome segregation during mitosis depends on the proper function
of specialized structural and cytoskeletal machinery. The duplicated
chromosomes are separated equally to daughter cells by the highly
dynamic fibers of the mitotic spindle called microtubules. The spindle
consists of a bipolar array of microtubules where the extreme ends of
the spindle are each anchored to a centrosome. Kinetochores are large
protein complexes that assemble onto centromeric DNA sequences and
physically attach the replicated chromosomes to the spindle fibers.
Ultimately, the maintenance of genomic integrity depends on the proper
attachment of chromosomes to the spindle and on the generation of
opposing tensile forces that pull the chromosomes apart. Failure in
either of these processes leads to unequal partitioning of the genome.
However, little is known about how chromatid-microtubule attachment
is mediated, or how opposing forces are generated. Currently, two anticancer agents, paclitaxel (taxol) and camptothecin, are used in the
treatment of various forms of the disease. Taxol promotes irreversible
polymerization of microtubules, disrupting their inherent dynamic nature.
Camptothecin functions by inhibiting topoisomerase I activity and leads
to large scale chromosome breakage when opposing tensile forces are
applied. The diverse action of these two anti-mitotic compounds and
the mechanical complexity of the segregation process suggest that many
protein components are involved. In fact, recent studies in more
genetically tractable organisms such as the budding yeast, S. cerevisiae,
have identified over 50 proteins involved in the chromatid-microtubule
attachment process alone and many of the functional orthologues have
yet to be identified in humans. Using a RNA library of 49,000 doublestranded (ds)RNA targeting approximately 24,000 genes, we performed
a loss-of-function screen for essential mitotic chromosome segregation
genes. We identified novel genes whose inactivation caused mitotic
arrest. Multi-parametric analysis of image-based data derived from a
high-content screen including phospho-histone H3 levels, cellular
proliferation and nuclear morphology allowed us to isolate both
checkpoint and independent segregation genes.
20
A HUMAN PROTEIN ATLAS FOR NORMAL AND
CANCER TISSUES
Mathias Uhlen1, Fredrik Ponten2
1
Stockholm, Sweden; 2Uppsala University, Uppsala, , Sweden
Alison L. Allan1, Michael Keeney2
1
University of Western Ontario, Oncology, Schulich School of
Medicine & Dentistry, London, Ontario, Canada; 2London Health
Sciences Centre, Hematology/Flow Cytometry, London, Ontario,
Canada
Given the multi-step nature of cancer development, there should be
several opportunities for therapeutic targeting of tumor cells and/or the
tumor microenvironment. The ideal way to identify and monitor disease
progression is through surrogate marker approaches that are minimally
invasive and allow for longitudinal testing, such as blood tests. Our
current research focuses on the development of such approaches, in
particular rare event detection by image and flow cytometric methods.
Identifying rare populations requires a different approach than standard
cytometry techniques, which rely mainly on positive and negative
decisions made in either one, or at most, two dimensional space. Recent
interest in identification and quantification of rare cell populations such
as circulating epithelial tumor cells and circulating endothelial cells in
cancer patients has pushed the limits of detection even further. Flow and
image cytometric methods are now being developed to identify cellular
populations with frequencies as low as 1-20 cells/ml. This presentation
will discuss the issues that must be addressed when designing an assay to
accurately detect rare populations of cells in blood or bone marrow,
with emphasis on detection of circulating endothelial cells and circulating
tumor cells in patients with solid tumors and in experimental mouse
models of cancer.
Antibody-based proteomics provides a powerful approach for the
functional study of the human proteome involving the systematic
generation of protein-specific affinity reagents. We have used this strategy
to construct a comprehensive, antibody-based protein atlas for expression
and localization profiles in 48 normal human tissues and 20 different
cancers (1). The Human Protein Atlas is publicly available
(www.proteinatlas.org) and contains, in the first version, approximately
400,000 high-resolution images corresponding to more than 700
antibodies towards human proteins. Each image has been annotated by
certified pathologists to provide a knowledge base for functional studies
and to allow queries about protein profiles in normal and disease tissues
(2). Our results suggest it should be possible to extend this analysis to
the majority of all human proteins thus providing a valuable tool for
medical and biological research, in particular for biomarker analysis in
various patient cohorts. Reference: (1) Uhlen M, Ponten F. (2005)
Antibody-based Proteomics for Human Tissue Profiling. Mol Cell
Proteomics 4(4): 384-393, (2) Uhlen et al (2005) A human protein atlas
for normal and cancer tissues, Mol Cell Proteomics, 4(12):1920-1932.
21
FLOW AND IMAGE CYTOMETRIC FRET FOR
DETECTING PROTEIN ASSOCIATIONS
János Szöllõsi1, György Vereb1, Gábor Horváth1, Péter Nagy1
1
University of Debrecen, Department of Biophysics and Cell
Biology, Medical and Health Science Center, Debrecen, , Hungary
Supramolecular organization of biomolecules at the cell surface or inside
the cell has an important role in determining the function and integrity
of cells. Specific techniques are available now for detecting molecular
proximity and interactions in cells, such as flow or image cytometric
93
variations of fluorescence resonance energy transfer (FRET). Flow
cytometric techniques offer the advantage of rapid analysis on a large
number of cells (~105 cells in some minutes) with a high statistical
accuracy and a possibility for analyzing heterogeneity at the population
level. Flow cytometry, however, does not provide any information
about the spatial localization of fluorescent probes, but instead measures
the fluorescence intensity averaged over each cell. In contrast,
microscopic techniques provide a high spatial resolution: conventional
fluorescence microscopies have a ~250 nm resolution limited by
diffraction of the optics. Although microscopies have several further
advantages in detecting molecular dynamics of changes in the
distribution or intensity of fluorescent probes, they suffer from a low
statistical reliability, especially in the case of quantitative measurements.
Thus, a combined application of flow and image cytometry in resolving
particular biological questions can be a very powerful approach. In
flow cytometry we applied fluorescent probes with longer wavelength
excitation and multiple wavelength detection in the emission regions so
that autofluorescence correction could be performed on a cell by cell
basis in FRET analysis. These facts improved the accuracy of the FRET
method and cells with low receptor expression, such as HPB-ALL cells
transfected with various CD45 isoforms were amenable to FRET
investigation. Combination of various forms of flow and image
cytometric FRET methods revealed distinctive expression and association
pattern of ErbB receptor tyrosine kinases on the surface of various
cancer cell lines sensitive or resistant to trastuzumab (Herceptin®).
Simultaneous application of image cytometric FRET methods based on
donor and acceptor photobleaching provided a useful dual FRET
approach revealing a unique coassociation pattern of integrins, CD44
and ErbB2 on the surface of tumor cells. By measuring the distances
between various monoclonal antibody epitopes on ErbB2 molecules
and the distances between epitopes and the cell membranes useful
information was provided for positioning the extracellular domain in
molecular modeling the nearly full length ErbB2 dimer. In this model
favorable dimerization interactions were predicted for the extracellular,
transmembrane and protein kinase domains, which may act in
coordinated fashion in ErbB2 homodimerization, and also in
heterodimers of ErbB2 with other members of ErbB family.
22
DO NOT MIND THE GAP: PROTEIN TRAFFICKING
BETWEEN THE ENDOPLASMIC RETICULUM AND THE
GOLGI APPARATUS IN PLANT CELLS
Federica Brandizzi1
1
University of Saskatchewan and Michigan State University,
Saskatoon/East Lansing, Saskatchewan/Michigan, Canada
Secretory materials are synthesized on the surface of the endoplasmic
reticulum (ER)1. They are then shipped from the ER to the Golgi
apparatus to be sorted either back to the ER or to distal secretory
compartments such as vacuoles and plasma membrane. It is vital for a
cell to regulate protein transport between these organelles. The ER and
Golgi are closely associated in plant cells2,3 (Fig. 1). How these two
organelles communicate with each other in plant cells is an important
question that remains largely unanswered. To provide further
understanding of the regulation of protein export from the plant ER, we
have explored the mechanisms of protein trafficking between the ER
and the Golgi apparatus using live cell imaging techniques. It appears
that plant cells contain multiple mobile Golgi stacks distributed over the
entire cytoplasm. These stacks move with the ER by means of actinmyosin motors3,4. The domains of the ER dedicated to the export of
proteins, the ER export sites (ERESs), form secretory units that move
along the surface of the ER together with the Golgi stacks4,5. We also
found that the integrity of Golgi and ERESs is regulated by the activity
of specific GTPases, such as Sar1 and Arf14,5. Finally, we determined
the existence of a stringent signal-regulated mechanisms for ER export
of multispanning, type I and type II membrane proteins6. For example,
we found that mutations of a specific di-acidic motif (DXE) in the
cytosolic tail of proteins such as CASP, a Golgi matrix protein with a
type II membrane topology7, cause a reduction of the export of this
protein from the ER6. ER export of type I and multispanning membrane
proteins appears to be similarly regulated6. Our results indicate that in
plant cells the ER and Golgi form a dynamic membrane system whose
components continuously cycle through the ER via a regulated membrane
trafficking pathway. References: 1. Nicchitta CV. Curr Opin Cell Biol
2002;14(4):412-6. 2. Boevink et al. Plant J 1998;15(3):441-7. 3.
Brandizzi et al. Plant Cell 2002;14(6):1293-309. 4.
daSilva et al. Plant
Cell 2004;16(7):1753-71. 5.
Stefano et al. Plant J 2006; in press. 6.
94
Hanton et al. Plant Cell 2005;17(11):3081-93. 7.
Mol Biol 2005;58(1):109-22.
Renna et al. Plant
Confocal image of a tobacco leaf epidermal cell transformed with
ERD2-YFP, a Golgi and ER marker3. Note that Golgi bodies
(arrow) are in close association with the ER network. Scale bar = 5
microns
Parallel Sessions
23
COHERENT ANTI-STOKES RAMAN SCATTERING
(CARS) MICROSCOPY FOR LABEL-FREE,
CHEMICALLY-SELECTIVE BIOMEDICAL IMAGING
Conor L. Evans1, Jeanette Kurian1, Eric O. Potma2, Mehron
Pourgish’Haag3, Daniel Cote3, Charles P. Lin3, X. Sunney
Xie1
1
Harvard University, Chemistry and Chemical Biology, Cambridge,
Massachusetts; 2University of California, Irvine, Chemistry, Irvine,
California; 3Massachusetts General Hospital, Wellman Center for
Photomedicine, Boston, Massachusetts
Advances in biomedical imaging have revolutionized our ability to
visualize living organisms at the cellular and sub-cellular levels. Despite
these developments, in vivo imaging with chemical contrast remains a
challenge as molecular selectivity typically requires the introduction of
specific labels. Many applications in biology and medicine would
significantly benefit from a noninvasive imaging technique that
circumvents such exogenous probes. In vivo microscopy based on
vibrational spectroscopic contrast offers a unique approach for
visualizing tissue architecture with chemical specificity. Coherent-antiStokes Raman scattering (CARS) microscopy is a non-linear imaging
technique capable of rapid vibrational imaging of thick biological
specimens. Backscattering of the intense forward propagating CARS
light in tissue gives rise to a surprisingly strong epi-CARS signal that
makes in vivo imaging possible. This substantial signal allows for realtime monitoring of dynamic processes, such as the diffusion of chemical
compounds, in tissues [Evans et al. PNAS 102, 16807 (2005)]. By
specifically tuning into the CH2 stretching vibrational frequency, we
demonstrate CARS imaging and spectroscopy of lipid rich tissue structures
in mouse skin, including sebaceous glands, corneocytes and adipocytes,
with unprecedented contrast at subcellular resolution.
24
QUANTITATIVE LIVE IMAGING DESCRIBES
MORPHOGENETIC NUCLEAR MOVEMENTS IN EARLY
DROSOPHILA EMBRYO
Cris Luengo1, Soile Keränen2, Charless Fowlkes3, Gunther
Weber4, Min-Yu Huang4, Oliver Rübel4, Bernd Hamann4,
Damir Sudar1, Jitendra Malik3, Mark D. Biggin2, David W.
Knowles1
1
Lawrence Berkeley National Laboratory, Life Sciences Division,
Berkeley, California; 2Lawrence Berkeley National Laboratory,
Genomics Division, Berkeley, California; 3University of California,
Berkeley, Computer Science Division, Berkeley, California;
4
University of California, Davis, Department of Computer Science,
Davis, California
The Berkeley Drosophila Transcription Network Project (bdtnp.lbl.gov)
is conducting a system-wide analysis of the transcription network in the
early Drosophila embryo. As part of this multidisciplinary effort, novel
imaging, image analysis and visualization methods have been developed
to construct the first three-dimensional (3D) atlas of gene expression
and morphology in an embryo at cellular resolution. Our aim is to
quantify the relative expression of hundreds of genes in wild type
embryos and in a series of mutant embryos, and to map these results
onto “stereotypical embryos”. Multiple-color in-situ hybridizations are
used to fluorescently label gene products of interest, and total DNA is
counter-stained. High resolution, multi-channel, 3D images are acquired
of entire embryos using two-photon excitation. Individual nuclei are
isolated from the DNA-stained images using novel, automated
segmentation techniques, and the relative gene expression within and
around each nucleus is then quantified. Novel techniques have been
developed to register data from many images onto a “stereotypical
embryo” and create exquisite quantitative visualizations of the data in
3D. One novel observation we have made is the systematic change in
nuclear packing densities during stage 5 (interphase cycle 14, up to
gastrulation), which can be seen by comparing fixed embryos of different
ages. To understand these nuclear movements, we have studied the
process in live histone2A-GFP embryos. Individual embryos were
imaged from the 13th cleavage cycle to the start of gastrulation in
approximately 3 minute intervals. Rapid temporal sequences were
important for tracking individual nuclei but restricted the imaging to
the portion of the embryo closest to the objective lens. Orientation of
the imaged embryo portions were determined by morphological features
during gastrulation and egg-length was determined from optical sections
taken through the middle of the embryo. These measurements allowed
images from multiple embryos to be combined into whole-embryomaps. The resulting patterns of nuclear packing density and flow-fields
correlate with the dorsal-ventral orientation of the embryo and the
future location of the ventral furrow. These results, which support our
fixed embryo work, show complex 3D nuclear movements prior to
gastrulation that will have to be considered to fully understand dynamics
in gene expression patterns.
25
ACQUIRING MULITPLE IMAGES OF LARGE TISSUE
SECTIONS IN BOTH FLUORESCENCE AND
TRANSMITTED LIGHT USING THE LASER SCANNING
CONFOCAL TISSUESCOPE
Trudey Nicklee1, David Hedley2
1
Toronto, Ontario, Canada; 2Princess Margaret Hospital, Toronto,
Ontario, Canada
Large tissue sections that overfill a microscope 10x objective field of
view typically must be imaged using a tiling method. In tiling, sequential
fields of view are acquired as separate image files. This is time consuming,
especially when algorithms are employed to align edges of individual
fields. Furthermore, depending on the quality of the stage movement
and algorithms used, artifacts may be introduced from misaligned fields.
The Biomedical Photometrics TISSUEscope (Waterloo, Ontario;
confocal.com) is a laser confocal scanning microscope, able to acquire
a low resolution image of an entire microscope slide as a single continuous
20 mm x 70 mm field in 30 seconds. Large subsections of interest can
then be imaged at selected resolution to 1µm per pixel. The instrument
is equipped with blue, green and red lasers, and can simultaneous acquire
images from two lasers. Software allows for the same selected field of
view to be repeatedly scanned with different excitation and emission
parameters. Therefore, multiple fluorescent images of this same field
of view are acquired aligned. This allows not only for quantification of
several markers in a single section, but also co-localization studies of
these markers. As well as allowing rapid image acquisition compared to
tiled field imaging at comparable resolution using a conventional
fluorescence microscope, the system also incorporates transmission
detectors that allow RGB imaging based on absorbance from the three
lasers. A fluorescently stained slide can therefore be imaged, then
restained and imaged for transmitted light using the same optical system.
This allows selected areas of interest to be identified by transmitted
light, then directly linked to the fluorescence images. Wide field
multispectral analysis of histological sections is a powerful technique
for studying complex molecular processes at the intact tissue level. The
laser scanning TISSUEscope is a novel instrument that offers several
advantages that we are currently evaluating. Applications include the
analysis of signaling pathways in sequential biopsies obtained from
patients in clinical trials of molecular cancer therapeutics, and studies
that address the problems of intratumoral heterogeneity.
26
COMPARISON OF FLUORESCENTLY AND CHROMATIC
LABELED TISSUE MICROARRAYS ANALYZED BY
LASER SCANNING CYTOMETRY
Ed Luther1
1
CompuCyte Corporation, Cambridge, Massachusetts
Tissue microarrays (TMAs) are becoming invaluable tools in biomarker
development. In developing automated analysis systems, the choice
between fluorescent or chromatic staining techniques is a primary
consideration. We have performed an evaluation comparing the two
staining techniques. Materials and Methods: TMAs consisting of 121
elements (0.5 mm cores) were stained with either fluorescent or
chromatic dyes and then submitted for blind analysis on a laser scanning
cytometry platform. Control samples and breast cancer tissues were
included. Serial sections of the arrays were stained with antibodies
against HER2/neu protein and counterstained for DNA, either with
chromatic reagents (DAB counterstained with hematoxylin) or
fluorescent reagents (Alexa Fluor 488 counterstained with propidium
iodide). The slides were analyzed on an iCyte® Automated Imaging
Cytometer. High-speed scans were used to locate individual core elements
within the array. High-resolution (0.25 micron) scans were then
performed on each element to obtain either fluorescence or laser light
absorption signals. Spectral deconvolution was used to separate the
HER2 and DNA staining. In the chromatically stained sections,
compensation techniques were applied for tissue autofluorescence.
Scanned images were segmented using both nuclear segmentation and
random sampling analysis. Results: In both TMA sets, the nuclear
segmentation and random segmentation techniques (chromatic R2 =
0.9017, fluorescence R2 = 0.8048) correlated well. The correlation
between the values from the chromatic and fluorescent labeled slides
was less satisfactory—R2 = 0.6595. To investigate the discordance,
analysis regions were defined around core elements falling into the
following categories, laser scan images of representative elements were
analyzed, and the staining patterns of the cores were confirmed. Category
+
Chromatic
Fluorescent Diagnosis 1 +
HER2/neu positive
+
2
HER2/neu positive – below fluorescence threshold 3
+
Autofluorescence artifacts 4 Negative Conclusions: Our
analysis showed greater sensitivity for TMAs stained with chromatic
dyes than for those with fluorescent dyes. This is contradictory to the
current belief that fluorescent dyes have greater quantitative capabilities
than chromatic IHC dyes, and is probably due to complications from
autofluorescence of the tissues. The ability to analyze both chromatic
and fluorescent TMAs on the same instrument platform allows
investigators great flexibility in selecting an appropriate staining
technology - fluorescence, chromatic or both.
95
27
HYPERCHROMATIC CYTOMETRY PRINCIPLES FOR
CYTOMICS BY SLIDE BASED CYTOMETRY
Attila Tárnok1, Mittag Anja1, Wiebke Laffers2, Dominik
Lenz3, Andreas Gerstner2
imaged. Thus, this endoscope advances the technology in this field by
delivering quality, high-resolution microscopic images with simultaneous
macroscopic location information.
Image Processing and Analysis 1
1
Cardiac Center, University of Leipzig, Research Laboratory,
Pediatric Cardiology, Leipzig, Saxony, Germany; 2University Bonn,
Bonn, Nordrhein-Westfalen, Germany; 3Purdue University, Purdue
University Cytometry Laboratories, West Lafayette, Indiana
Multicolor and polychromatic analysis of biological specimens has
become increasingly important due to the emerging new fields of highcontent and high-throughput single cell analysis for Systems Biology
and Cytomics. Combining different technologies and staining methods
polychromatic analysis can be pushed further forward to virtually
measure anything stainable in a cell. We termed this approach
hyperchromatic and present different components suitable to be
combined for achieving this task. For cell analysis Slide Based Cytometry
(SBC) technologies are ideal as, unlike flow cytometry, it is a nonconsumptive method, i.e. the analyzed sample is fixed on the slide and
can following manipulation of the object be subsequently reanalyzed.
In this overview we demonstrate on a SBC instrument, the Laser
Scanning Cytometer, various approaches for hyperchromatic analysis.
The different components demonstrated here are: 1) polychromatic
cytometry (staining of the specimen with eight or more different
fluorochromes simultaneously), 2) iterative restaining (using the same
fluorochrome for restaining and subsequent reanalysis), 3) differential
photobleaching (differentiating fluorochromes by their different
photostability), 4) photoactivation (activating fluorescent nanoparticles
or photocaged dyes), and 5) photodestruction (destruction of FRET
dyes). Based on the feature of relocating cells that are immobilized on
a microscope slide the identical cells can be subsequently reanalyzed
and the data collected on the single cell level after manipulation steps.
With the intelligent combination of several different techniques
hyperchromatic cytometry allows to quantify and analyze virtually all
components of relevance on the identical cell. The information gained
per specimen is only limited by the number of available antibodies and
by sterical hindrance.
28
A MULTIMODE ENDOSCOPE FOR SIMULTANEOUS
MACROSCOPIC AND MICROSCOPIC IMAGING OF
TISSUES IN VIVO
29
TOPOLOGY PRESERVING STACS SEGMENTATION OF
PROTEIN SUBCELLULAR LOCATION IMAGES
Amina Chebira1, Gowri Srinivasa1, Lionel Coulot2, Heather
Kirschner3, Jose M. F. Moura3, Jelena Kovacevic1, Elvira
Garcia Osuna4, Robert F. Murphy5
1
Carnegie Mellon University, Biomedical Engineering, Carnegie
Institute of Technology, Pittsburgh, Pennsylvania; 2EPFL, Lausanne,
Switzerland; 3Carnegie Mellon University, Electrical and Computer
Engineering, Pittsburgh, Pennsylvania; 4Carnegie Mellon
University, Biomedical Engineering, Pittsburgh, Pennsylvania;
5
Carnegie Mellon University, Biological Sciences and Biomedical
Engineering, Pittsburgh, Pennsylvania
We present an algorithm for the segmentation of multicell
fluorescence microscopy images. Such images abound and a
segmentation algorithm robust to different experimental conditions as
well as cell types is becoming a necessity. In cellular imaging, among
the most often used segmentation algorithms is seeded watershed.
One of its features is that it tends to oversegment, splitting the cells, as
well as create segmented regions much larger than a true cell. This
can be an advantage (the entire cell is within the region) as well as a
disadvantage (a large amount of background noise is included). We
present an algorithm which segments with tight contours by building
upon an active contour algorithm—STACS proposed by
Pluempitiwiriyawej at al. We adapt the algorithm to suit the needs of
our data and use another technique, topology preservation proposed
by Han et al, to build our topology preserving STACS (TPSTACS).
Our algorithm significantly outperforms the seeded watershed both
visually (see Figure, seeded watershed on the left, TPSTACS on the
right) as well as by standard measures of segmentation quality (see
Table).
Silas J. Leavesley1, Bartlomiej Rajwa2, J. Paul Robinson3
1
Purdue University, Biomedical Engineering, Weldon School of
Biomedical Engineering, West Lafayette, Indiana; 2Purdue
University, Basic Medical Sciences, Veterinary Medicine, West
Lafayette, Indiana; 3Purdue University, Basic Medical Sciences &
Biomedical Engineering, West Lafayette, Indiana
Diagnosis of pathologies often includes gathering information using
various diagnostic medical imaging modalities, and as a result of this
information, the possible biopsy of tissues for further analysis. In many
cases, the region(s) in which to perform a biopsy are detected and
delineated by diagnostic images. Pathologies of the alimentary,
respiratory, and urinary systems often make use of endoscopic imaging
modes (mainly traditional and fluorescence endoscopy) to detect
potential lesions, and use biopsy as a method for confirming the nature
of the detected lesion. Thus, increasing the sensitivity of endoscopic
diagnosis will aid in correctly delineating the boundaries of detected
lesions, and increasing the specificity of endoscopic techniques may
help to decrease the total number of biopsies taken.
This work
outlines the development of a new type of endoscope that features a
forward-facing macroscopic video imaging port, and a side-facing
microscopic imaging port. Both of these modes of imaging can operate
simultaneously, and both can be operated in reflected light and
fluorescence modes, allowing a physician to “zoom-in” on potential
lesions and inspect their cellular makeup. The ability to inspect these
suspected areas should decrease the number of false positive diagnoses,
which could serve to decrease the number of biopsies, in many cases.
This is appealing for two reasons, first it will serve to reduce tissue
damage and pain to patients; second, it will reduce some costs associated
with unnecessary biopsies. In addition, with the advanced software
used in this system, the physician has greater control over what is actually
96
SW [%] TPSTACS [%]
30.8
80.5
Area Similarity (AS)
Area Overlap (AO)
Recall (R)
Precision (P)
HS
DNA
HS (T=70%)
DNA (T=95%)
HS (T=70%)
DNA (T=95%)
62.2
62.3
37.9
36.8
40.0
36.3
82.1
99.8
71.1
99.1
76.8
99.1
30
TWO AND THREE DIMENSIONAL SEGMENTATION OF
WHOLE CELLS AND CELL NUCLEI IN TISSUE
Dean P McCullough1, Daniel Baggett2, Stephen J. Lockett3
1
National Cancer Institute, Advanced Biomedical Computing Center,
Frederick, Maryland; 2Worcester Polytechnic Institute, Mechanical
Engineering, Worcester, Massachusetts; 3NCI / SAIC-Frederick,
Frederick, Maryland
Communications between neighboring cells in large part drive tissue
development and function, as well as disease-related processes such as
tumorigenesis. In order to understand the molecular basis of these
processes, it is necessary to quantitatively analyze specific molecules in
adjacent individual cells or cell nuclei of the intact tissue. A major
bottleneck preventing widespread use of such analyses is the lack of an
efficient method that assures correct segmentation of all individual,
whole cells in a given intact tissue volume of interest from 3D images.
Consequently, we have developed software for identifying the optimum
border around each individual cell or cell nucleus, a process known as
segmentation, from 2D and 3D microscope images of intact tissue labeled
with a fluorescent cell or nuclear surface marker. In the 2D case the
optimum border was defined as the border that has an average intensity
per unit length greater that any other possible border around the same
cell or nucleus. Implementation of the algorithm required the user to
indicate two points for each cell, one inside the cell and the other on the
border. Thereafter segmentation was automatic and was peformed using
dynamic programming. The method correctly detected virtually 100%
of cells, because determination of the optimum path is not significantly
affected by intermittent labeling of the cell borders, by diffuse borders,
or by spurious signals away from the borders. It also contains interactive
tools, which allows subsequent visualization and correction of the image.
The method is also highly efficient due to only minimal user interaction.
We have extended this method to 3D segmentation, which begins with
2D segmentation in a user-selected plane approximately through the
center of the cell. Then the automatic algorithm separately finds the
two surfaces of the cell in the planes above and below the user selected
plane using dynamic programming. The algorithm does not find the
true optimal surface since this is too computationally expensive, but
closely approximates the optimum by finding a succession of partially
optimal surfaces using the greedy algorithm with a look ahead of n
steps. Following segmentation, the user may add points that are required
to be on the surface to correct any perceived errors. The algorithm has
been tested on a wide variety of biological tissue samples and will
segment moderately irregularly shaped cells containing concavities.
Work performed under contract grant sponsor: NCI/NIH; Contract #:
NO1-CO56000.
31
A GENERAL TECHNIQUE FOR SEGMENTATION OF
Zachary Pincus1, Julie A Theriot2
1
Stanford University, Biomedical Informatics, School of Medicine,
Stanford, California; 2Stanford University, Biochemistry, School of
Many ad hoc methods exist for separating cells from the image
background (and, with less success, from each other) in light
micrographs. Here we present a general method that is applicable to
many cell types and imaging modalities. A persistent difficulty with cell
segmentation tools is that assumptions about cell shape, imaging
conditions, and cell packing are “hard-coded” into the cell-finding
logic. We instead make these assumptions explicit and modular. By
using a parameterized “shape model” learned from training examples,
our tool makes no implicit assumptions about cell shape. Performing
the principal components analysis on cell outlines provided by the user
produces a set of linearly independent modes of shape variation (e.g.
large vs. small, round vs. elongated, etc.). These shape modes can then
be recombined to generate candidate cells from a distribution of expected
shapes. Because the only input required is a small set of shapes, it is easy
to reconfigure this module for different cell types. We free our method
from assumptions about the relationship between pixel values and the
presence or absence of cells by converting images into a canonical
form. Specifically, we use machine learning tools to convert images
into “probability maps” where the value of each pixel is the likelihood
that that pixel is inside of a cell. Simple k-nearest-neighbor classification
of pixels based on local texture statistics is sufficient to transform images
of many different types (e.g. phase contrast, DIC, epifluorescence) into
simple-to-interpret probability maps. The only required inputs are a
few image regions which have been manually labeled as “cells” or
“background;” thus this module is also easy to reconfigure for different
imaging conditions. To find a single cell in an image, a shape model is
iteratively fit to a probability map. We numerically optimize the
parameters of a given shape model (creating different candidate shapes)
and its pose (x- and y-position and rotation) to maximize a goodnessof-fit function which evaluates how well the model fits to the probability
map. This function evaluates the prior likelihood of the candidate shape,
the distance between candidate shape edges and image edges, and the
fraction of pixels with low likelihood of being in a cell that are
nevertheless inside the candidate shape. We then refine the initial fit by
deforming the shape to better fit the image within a level-set framework.
This process is repeated to find all cells in an image. This method has
shown human-competitive results in separating out individual cells from
the background and from each other on many different image and cell
types, including fixed S2 cell cultures, growing bacterial mats, and
membrane-stained Drosophila epithelia.
32
FILO: AN UNBIASED SPATIAL ANALYSIS OF FISH
SIGNALS IN INTERPHASE NUCLEI
Prabhakar Reddy Gudla1, Addison Z Yee2, Takumi
Takizawa3, Tom Misteli3, Stephen J. Lockett1
1
NCI/SAIC-Frederick, Image Analysis Lab, Frederick, Maryland;
NCI-Frederick, Image Analysis Lab, Frederick, Maryland; 3NCI,
Cell Biology of Gene Expression, Bethesda, Maryland
2
Spatial organization of interphase nuclei into well defined compartments
and the positioning of gene sequences in interphase nuclei significantly
impacts protein expression and cell function. Such phenomena are
discovered through the application of statistical methods that analyze
the non randomness of spatial distributions of fluorescence in situ
hybridization (FISH) labeled sequences in terms of their distance to the
nuclear centers and their proximity to each other. We have developed
an algorithm (FILO) that uses automatic fuzzy-C-means clustering to
segment cell nuclei and FISH signals from 2D images. Using statistical
modeling of the FISH copy number in diploid cells, the algorithm
found approximately 95% of true signals (tested using labeled protooncogene MYC and immunoglobulin-ë genes in human lymphoblast
cells) and approximately 6% of detected signals were considered spurious.
These results closely matched 96% and 7%, respectively, from visual
analysis, thus strongly validating the segmentation procedure. We have
developed novel, non-parametric spatial statistical tests, applicable for
elliptically-shaped nuclei, which independently analyze the proximity
of each FISH signal to the center of nuclei, the proximity of each signal
to the major axis of the nucleus and the proximity of homologous and
heterologous pairs of signals to each other. For example, the test will
determine whether the proximity of FISH signals to each other is solely
explained by their proximity to the nuclear center. This is accomplished
by comparing the distribution of distances between pairs of FISH signals
to the distribution of distances between pairs of random points using the
Kolmogorov-Smirnov test, where the random points are conditioned to
have the same distribution of distances to the nuclear centers as the
experimental FISH signals. We call this approach: “bias-correction”.
The test has been validated by simulation, applied to actual samples and
generalizes to the independent analysis of many spatial parameters
relevant to the genomic organization of the nucleus. Ongoing
developments include extension to analyze FISH signals in arbitraryshaped nuclei. This involves application of the distance transform to
calculate the probability that each FISH signal is close to the edge of its
nucleus, and then combining these probabilities into an overall
probability for the population of nuclei using Fisher´s Chi-Square
method. Overall, “FILO” will be an important tool for analyzing the
complex genetic organization of interphase nuclei, including the potential
for analysis of 3D images of polarized nuclei in tissue and for high
throughput analysis. Acknowledgments: Work performed under contract
grant sponsor: NCI/NIH; Contract #: NO1-CO56000
97
33
IMAGE ANALYSIS AND METADATA PROCESSING FOR
CELL STRUCTURE AND FUNCTION DESCRIPTION.
EXAMPLES OF APPLICATION AS A NEW DIAGNOSTIC
APPROACH IN THREE DIFFERENT FIELDS:
HAEMATOLOGY, TOXICOLOGY AND MICROBIOLOGY
Maria Cristina Albertini1, Marco Rocchi2, Augusto Accorsi1,
Laura Teodori3
1
University of Urbino, Institute of Biochemistry C.Fornaini, Urbino,
Italy; 2University of Urbino, Institute of Biomedical Engineering,
Urbino, Italy; 3ENEA CR CASACCIA, BIOTEC-MED, Rome,
Italy
In order to describe biological states and processes, the collection and
the evaluation of a large amount of image data are becoming more and
more important. Consequently, the search for suitable complex statistics
and the development of algorithms, allowing us to model the data and
compile the knowledge gained from bio-imaging is the next challenge.
We used a platform of statistical methods such as: multivariate statistics
(PCA, discriminant analysis and multidimensional scaling) circular
statistics (analysis of directional data) and shape analysis, to express
analytical results from captured meaningful cell image information
acquired from optical, electron and atomic force microscopy. All these
microscopies have added high-resolution spatial dimensions information
that enabled us to quantitatively detect cell orientation, surface properties
and metabolic features. We reported examples of application to three
different research areas: haematology, microbiology and toxicology.
By this approach we were able to: i) diagnose some shape related RBC
pathologies based on morphological automated analysis (essential
hypertension prediction); ii) evaluate water Vibrio alginolyticus
contamination, distinguishing between viable-but-not-culturable-cells
to culturable-cells; iii) quantitatively analyse physiological and
morphological alterations from static magnetic field exposure,
demonstrating altered cell shape, diverse orientation and abnormal
membrane structure; iv) increase the power of the comet assay DNA
damage evaluation, distinguishing different DNA damages from
chemical/physical noxia. In conclusion, the results demonstrated that
through combining the imaging of cells with powerful image analysis
algorithms, one can acquire a deeper knowledge on multiple biochemical
and morphological pathways at the single-cell level to be used as a
diagnostic tool.
34
NONINVASIVE FORWARD-SCATTERING SYSTEM FOR
RAPID DETECTION, CHARACTERIZATION, AND
IDENTIFICATION OF LISTERIA COLONIES. IMAGEPROCESSING AND ANALYSIS
Bulent Bayraktar1, Padmapriya P Banada2, J. Paul
Robinson3, Arun K. Bhunia2, E. Daniel Hirleman4,
Bartlomiej Rajwa3
1
Purdue University, Electrical and Computer Engineering, Schools
of Engineering, West Lafayette, Indiana; 2Purdue University,
Molecular Food Microbiology Laboratory, Department of Food
Science, West Lafayette, Indiana; 3Purdue University, Basic Medical
Sciences, Veterinary Medicine, West Lafayette, Indiana; 4Purdue
University, School of Mechanical Engineering, Engineering, West
Lafayette, Indiana
Bacterial contamination by Listeria monocytogenes not only puts the
public at risk but also is costly for the food-processing industry.
Traditional biological and chemical methods for pathogen identification
require complicated sample preparation for reliable results. Optical
scattering technology has been used for identification of bacterial cells
in suspension, but with only limited success. Extracellular materials
produced by a culture in a colony along with cellular arrangements
may provide unique signatures that are necessary for differentiation of
colonies by light scattering. We have developed a noninvasive optical
forward-scattering system for rapid detection, characterization, and
identification of Listeria colonies grown on solid surfaces. The presented
work includes application of computer vision and pattern recognition
techniques. Bacterial colonies with a diameter of approximately 1.8 to
1.9 mm and a thickness of around 0.3 to 0.4 mm (measured along the
optical axis) were analyzed with a laser scatterometer. Circular scatter
patterns formed by bacterial colonies illuminated by red laser light were
analyzed using Zernike (continous) or Tchebichef (discrete) moment
98
invariants. Discrete moments do not possess the discretization errors
inherent in continuous moments. This important advantage allows better
fulfillment of orthogonality and invariance properties. Use of discrete
moments improves the speed of our image processing algorithms. This
is attained by calculating discrete polynomial coefficients directly from
their definitions employing arbitrary precision arithmetic, rather than
using recurrence relationships. The constructed coefficients are stored
in look-up tables, and retrieved during the process of image analysis.
This approach also eliminates a potential of numerical instability due to
inadequate numeric precision. Principal component analysis and
hierarchical clustering were performed on the results of feature extraction
for colony differentiation. Classifications using linear discriminant
analysis, partial least squares, and neural networks were used to test the
feasibility of automated determination of bacteria pathogenicity on the
basis of colony scatter patterns. The overall system is robust and can be
extended into an automated user-friendly device for detection and
differentiation of various pathogenic bacteria.
35
ENHANCED DIFFERENTIATION MODULE (EDM) FOR
CLINICAL FLOW CYTOMETRY ANALYSIS
J. Paul Robinson1, Kathy Ragheb2, Cheryl Holdman3, Valeri
Patsekin4, Christakis Christodoulou5, Bill Kiroviac6, Paul
Church7, Todd Lary7
1
Purdue University, Basic Medical Sciences & Biomedical
Engineering, West Lafayette, Indiana; 2Purdue Unviersity, Basic
Medical Sciences, West Lafayette, Indiana; 3W. Lafayette, Indiana;
4
Purdue University, W. Lafayette, Indiana; 5Beckman-Coulter,
Engineering, Physical Sciences and Engineering, Miami, Florida;
6
Beckman-Coulter, Miami, Florida; 7Miami, Florida
We report on a new technology for flow cytometry to improve the light
scatter collection of a routine instrument. The Enhanced Differentiation
Module (EDM) developed for the FC500 and Altra flow cytometers
provides several new scatter options for routine flow cytometry. Forward
Angle Light Scatter (FALS) has been a standard feature of almost all
flow cytometers for over 30 years. While inital developments in the
field included studies of many angles of scatter, almost all instruments
adopt a standard 2-4 degree scatter detection angle. The EDM module
developed by Beckman-Coulter uses a fiber optic array with multiple
rings at preset angles to collect significantly enhanced scatter signatures.
Inital tests of the EDM were performed on a standard 2 laser FC500
benchtop cytometer. In the study, two identical instruments were run
side-by-side with one instrument modified to accept the EDM. We present
data showing the imact of using the different angles of scatter on very
small beads, blood cells and regular cell cultured in flasks with a variety
of treatements. The EDM as installed allows the use of individual or
combined collection rings in addition to the implementation of neutral
density filters to reduce the signal intensity on the detector if necessary.
A very useful feature of the device was the ability to modify the gains
on each detector to achieve optimal differentiation for any particular
sample type. Current flow cytometry essentially assumes that a single
forward scatter signal is adequate for most use. We have shown that this
is seriously mistaken perception. Multiple scatter angles undoubtedly
allow for differentiation of morphologically distinct populations
otherwise inseparable by a standard scatter detector. We believe that this
modification of a routine instrument will open new opportunities for
cellular differentiation for malignant cells, cells which have phagocytosed
particles, minor size changes and other refractive index alterations that
differentiate cells or particles.
36
SORTING OF ULTRA-SMALL VESICLES FOR
PROTEOMIC ANALYSIS
James N. Higginbotham1, Zheng Cao2, Thomas J. Utley3,
James E. Crowe4, Robert J. Coffey5
1
Vanderbilt University, Pediatric Infectious Diseases, Vanderbilt
University Medical Center, Nashville, Tennessee; 2Vanderbilt
University, Medicine and Cell and Developmental Biology,
Nashville, Tennessee; 3Vanderbilt University, Microbiology and
Immunology, Vanderbilt University Medical Center, Nashville,
Tennessee; 4Vanderbilt University, Medical Center, Nashville,
Tennessee; 5Vanderbilt University, Medicine, School of Medicine,
Nashville, Tennessee
Commercial flow cytometers have undergone recent design
improvements in the optical collection and electrical signal processing
that have dramatically improved the ease of operation and capabilities
of sorting cytometers. We have recently customized a BD FACSAria
with the addition of a forward scatter PMT that allows improved resolution
of particles smaller than 200 nm in diameter. In this study we report
methods that allow the purification of exocytic vesicles ranging in size
from 30-200 nm to greater than 98% purity. Briefly, Naked-2-GFP
fusion containing vesicles are partially purified by ultracentrifugation
to approximately 60 % purity and counter stained with DID. Subsequent
sorting proteomic analysis generated several tags of importance with
regard to vesicle trafficking and functional binding of TGF-Ñ, the
ligand of Naked-2. Application of this work to other types of vesicles is
currently underway. Finally, these developments have been applied to
the detection of cell-free GFP-tagged human rotavirus virus-like particles
that are less than 100 nm in size.
37
A NOVEL SOFTWARE ARCHITECTURE THAT ALLOWS
THE USE OF WINLIST FOR REAL-TIME DATA
ACQUISITION AND SORTING CONTROL OF A HIGHLY
AUTOMATED PARALLEL SORTING (HAPS)
Gary Durack1, Mark Hubbard1, Jeremy Hatcher1, C. Bruce
Bagwell2
1
iCyt Visionary Bioscience, Champaign, Illinois; 2Verity Software
House, Inc., Topsham, Maine
A promising new approach for ultra high-throughput cell sorting is the
use of Highly Automated Parallel Sorting (HAPS) systems. In these
systems, many HAPS channels can be operated in parallel to achieve a
throughput of millions of cells per second. We have developed and will
demonstrate a standard interface to the iCyt HAPS system that allows
the use of Verity Software´s WinList listmode analysis software as the
primary graphical user interface (GUI) for real-time data acquisition,
definition of cell sorting regions, definition of sort logic and listmode
data analysis. WinList, a very advanced data analysis package, was
modified by Verity Software to utilize the iCyt instrument interface. On
the iCyt system, the individual HAPS channels are controlled by an
embedded single board computer (SBC). Once programmed, each HAPS
channel will perform all sort operations independent of external control.
The SBC communicates with external computer applications such as
WinList via TCP/IP over a local area network. This allows multiple
computers to send and receive data from the SBC. The interface allows
data streams to pass in real-time from the SBC to any computers on the
LAN running WinList. The WinList computers send sort region, sort
logic, and control information to the SBC. The system allows data from
multiple HAPS channels to be displayed in a single instance of WinList.
It also allows multiple instances of WinList to view data from the same
HAPS channel. This architecture is scalable and can be used to support
control of any number of HAPS channels. The advanced data processing
and control architecture presented is an incremental step toward realizing
ultra high-throughput cell sorting in highly parallelized systems.
38
CLASSIFYING CELL SIGNALING PROFILES IN
LEUKEMIA
Nikesh Kotecha1, Jonathan Michael Irish2, Garry P. Nolan3
1
Stanford, California; 2Stanford University, Oncology Division,
School of Medicine, Stanford, California; 3Stanford University,
Molec Pharmacology, School of Medicine, Stanford, California
Most human diseases involve abnormal cell signaling behavior ranging
from cells being exposed to an abnormal amount of signals to a
malfunction in cell mechanism (e.g.cancer). Recent advances in antibody
and flow cytometry technologies have led to the ability of using FACS
to measure intracellular changes within cell populations[1,2]. These
changes are often transient (e.g. phosphorylations) and are the
mechanisms by which tumor cells achieve malignant behavior and
manipulate their environment. We have used this technique to discover
new and alternate signaling mechanisms[3,4] as well as show its use as
a novel way of profiling cancer patients[5]. Analysis techniques for the
considerable amount of data generated (e.g. 50,000 cells/sample x 6
measurements/cell) however require human intervention to determine
relevant cell subsets prior to sample comparison and ignore potentially
informative data present in small subsets of cells defined in
multidimensional signaling space. We have developed an approach based
on principal component analysis (PCA) for comparing samples without
subset identification. In particular, we have applied it to classify acute
myeloid leukemia (AML) samples from Irish et al[5] and can (1) rank
or cluster samples and (2) extract distinguishing features/cell subsets
between samples. Identification of such signaling subsets can be useful
in the discovery of small populations of cancer cells responsible for
poor clinical outcome (e.g. cancer stem cells). References: 1. Krutzik
PO, Irish JM, Nolan GP, Perez OD. Analysis of Protein Phosphorylation
and Cellular Signaling Events by Flow Cytometry: Techniques and
Clinical Applications. Clin Immunol. 2004 Mar;110(3):206-21. 2.
Krutzik PO, Nolan GP. Intracellular Phospho-Protein Staining Techniques
for Flow Cytometry: Monitoring Single Cell Signaling Events. Cytometry
A. 2003 Oct;55(2):61-70. 3. Sachs K, Perez O, Pe’er D, Lauffenburger
DA, Nolan GP. Causal Protein-Signaling Networks Derived from
Multiparameter Single-Cell Data. Science. 2005 Apr 22;308(5721):5239. 4. Perez OD, Krutzik PO, Nolan GP. Flow Cytometric Analysis of
Kinase Signaling Cascades. Methods Mol Biol. 2004;263:67-94. 5.
Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT,
Nolan GP. Single Cell Profiling of Potentiated Phospho-Protein Networks
in Cancer Cells. Cell. 2004 Jul 23;118(2):217-28.
39
CLUSTER AND PRINCIPAL COMPONENT ANALYSIS
FOR THE IDENTIFICATION OF COMPLEX
PHENOTYPES
Enrico Lugli1, Marcello Pinti1, Leonarda Troiano1, Milena
Nasi1, J. Paul Robinson2, Valery Patsekine2, Caterina
Durante3, Gianfranco Salvioli4, Marina Cocchi3, Andrea
Cossarizza1
1
University of Modena and Reggio Emilia, Dept. of Biomedical
Sciences, Modena, Italy; 2Purdue University, Basic Medical
Sciences, West Lafayette, Indiana; 3University of Modena and
Reggio Emilia, Dept. of Chemistry, Modena, Italy; 4University of
Modena and Reggio Emilia, Dept. of Gerontology, Modena, Italy
Polychromatic flow cytometry allows the determination of multiple
antigens in the same cell and permits, at least in part, the elucidation of
complex networks in the immune system. However, the main problem
of this powerful technology remains the analysis of the data, as multiple
determinations at single cell level can identify a high number of
populations, which obviously results from the combination of antigens
(typically, 2 to the n, where n is the number of fluorescent parameters).
Here we propose the use of cluster analysis and principal component
analyses (PCA) in order to simplify >7 colour data visualization. We
studied the expression of antigens involved in the differentiation
(CD45RA and CCR7), survival (CD127) and activation (CD95 and
CD38) of CD4+ and CD8 + T lymphocytes from subjects with different
age (young and middle-age donors and centenarians) and we analyzed
all possible populations by combining the expression (positivity or
negativity, as well as intermediate - dim - expression) of these molecules.
A 16-parameters CyFlow ML (Partec GmbH, Muenster, Germany),
99
equipped with a blue solid state laser (488 nm, 200 mW), a UV Mercury
lamp HBO (100 long life, 100 W), a red diode laser (635 nm, 25 mW),
a green solid state laser (532 nm, 50 mW) and a CCD camera was used.
Cluster analysis identified T cell dynamics occurring in immunological
aging: loss of naïve cells with accumulation of memory cells and, in
particular, increase of subsets characterised by tendency to effector
phenotype (CD95 + CD127-). PCA was able to cluster donors with
different age on the basis of flow cytometric profile and, in particular,
to identify T cell subsets that best characterise a specific group of subjects
and that most contribute to the total variance of the entire system. This
led us to determine that CD4+ and CD8+ T cells from young donors were
mainly composed by CD45RA+CCR7 + naïve T cells expressing CD127
and CD38 but lacking CD95 while centenarians displayed the presence
of central (CD45RA-CCR7+) and effector memory (CD45RA-CCR7-)
populations in CD4+ T cell compartment and preferential expansion of
the CD127-CD95+CD38- effector memory subset in CD8+ T cells. Our
data indicate that bioinformatic softwares could be used to analyze
multicolor flow cytometric data and to simply identify groups of subjects
on the basis of cellular subsets obtained by combination of antigens.
40
BIOINFORMATICS DATA STANDARDS FOR FLOW
CYTOMETRY
Ryan R. Brinkman1, Josef Spidlen1, Adam S. Treister2,
Clayton Smith1, Michael B. Ochs3, Robert Gentleman4,
Charles Schmitt5, Perry Haaland5
1
British Columbia Cancer Research Centre, Terry Fox Laboratory,
Vancouver, British Columbia, Canada; 2Tree Star, Inc., Ashland,
Oregon; 3Fox Chase Cancer Center, Philadelphia, Pennsylvania;
4
Fred Hutchinson Cancer Reserch Center, Seattle, Washington; 5BD
Technologies, Research Triangle Park, North Carolina
We are developing free, open source and commercial software compatible
bioinformatics standards and software tools for flow cytometry. The
scope of this project includes development of an ontology, a data base
schema, statistical and visual analysis tools, and a gating standard in the
Extensible Markup Language (XML). The gating standard is presently
our most well-developed component. Gating in flow cytometry is a
process for defining a boundary that delineates the characteristics of
particles for further analysis or sorting. During gating users define
regions around a group of events of interest and then choose to include
or exclude these events using some form of Boolean logic. Although
flow cytometry has a successful data format standard, there is no shared
representation of gates. This prevents a variety of collaborative
opportunities to recreate experimental methods and results. We have
developed a proposal on how to form gate definitions that can facilitate
the interchange and validation of data between different software
packages, and provide the means to better integrate methods with results
in reporting. Specifically, we have developed a detailed description of
the gating specification, a W3C Schema usable to validate XML
documents, user documentation, and a set of examples of gating XML
files. A software tool that implements the specification includes
functionality to read a Flow Cytometry Standard data file, an
accompanying XML file which describes the gates of interest, and the
ability to process this information to provide descriptive statistics.
Robustness problems are especially serious in geometric computation,
since numerical errors can propagate into the combinatorial computations
and result in complete failure of the algorithm. Our example
implementation uses algorithms which are based on a user-definable
precision model and contains code for robust geometric computation.
As a result, the software tool will be useful to validate compliance of
alternative software tools that implement the gating standard. The gating
specification currently supports gating in n dimensions, including
rectangular gates (e.g., range gates, 3D box regions, and n-dimensional
hyper-rectangular regions), polygon gates, ellipsoid gates, decision tree
structures and Boolean collections of the any of the types of gates.
Support for platform independent validation of 2D gates is available
from the JTS Topology Suite, and support for higher dimension polytopes
through platform specific implementations based on Qhull.
100
Cell Physiology 1
41
PLASTICITY OF THE TUMOUR CELL GLYCOCALYX
Paul J Smith1, Emeline Furon1, Sally Chappell1, Marie
Wiltshire1, Robert A Falconer2, Laurence Patterson2, Andrew
Goater3, David Morris3, Julian PH Burt3, Rachel J. Errington4
1
Cardiff University, Cardiff, Wales, United Kingdom; 2Bradford
University, Institute of Cancer Therapeutics, Bradford, West
Yorkshire, United Kingdom; 3University of Wales, Bangor, Institute
of Bioelectronic and Molecular Microsystems, Bangor, Gwynedd,
United Kingdom; 4Cardiff University, Medical Biochemistry,
Medicine, Cardiff, Wales, United Kingdom
Phenotypic plasticity (PP) in tumour cell populations has the undesirable
potential to maximise Darwinian fitness for the selection of metastatic
and drug-resistant variants. Changes in surface carbohydrate expression
have been implicated in this process - the surface glycocalyx altering
cell behaviour within a multicellular environment and assisting metastatic
spread through enhanced cell detachment. Polysialic acid (PSA),
providing polyanionic cell-surface decoration of the neural cell adhesion
molecule (NCAM), is thought to be associated with the property of the
early dissemination of cells from the primary tumour mass of small cell
lung cancer (SCLC) - a common metastatic cancer expressing
chemoresistance. Our aim was to explore the plasticity of the glycocalyx,
and NCAM decoration in particular, together with the impact on gene
expression and cellular dielectric properties. We have used a SCLC
model (NCI-H69) and substrate adhesion as a selective principle to
provide extremes of reversible phenotypic behaviour reflecting adherent
versus non-adherent growth. We found reduced PSA-decoration of
NCAM in adherent cells using flow cytometry and confocal imaging.
PSA decoration could be re-gained by the growth of adherent cells on
non-attaching hydrophobic surfaces without any induction of cell death.
PSA reduction was accompanied by a downregulation of the critical
glycolytic pathway genes GNE and FUT8, rather than modulation of
specific polysialyltransferase genes. The reductions in NCAM decoration
observed in vitro were re-iterated when cells were grown as mouse
xenografts. The in vitro changes were accompanied by an increase in
cytoplasmic membrane permittivity, but no change in zeta potential, for
adherent cells as determined by electrokinetic analysis and multishell
modelling. We outline a new probeless approach analysing the metastatic
phenotype for diagnostics and drug screening purposes. There was no
evidence that PP acted to modify pathways for cell cycle control, multidrug resistance or tumour growth potential per se. However, cDNA
microarray analysis revealed that genes modulated during acquisition
of the adherence phenotype were linked through VEGF, with significant
down-regulation of pro-metastasis, angiogenesis and ECM-related genes.
Furthermore, the most prominent proteoglycan expression gained upon
acquisition of adherence was glypican-3 - the encoding gene GPC3
being a potential tumour suppressor in SCLC. We conclude that PP can
reversibly generate cellular phenotypes with elevated metastatic potential
without recruiting de novo drug resistance - providing new targets for
therapeutic control.
42
LIPID RAFTS REGULATE PDGFR CLUSTERING,
ACTIVATION, INACTIVATION AND DOWNSTREAM
SIGNALING IN A CELL CONFLUENCE-DEPENDENT
MANNER
György Vereb1, László Ujlaky-Nagy1, János Szöllõsi1
1
University of Debrecen Medical and Health Science Center,
Debrecen, Hungary
PDGF receptors are transmembrane tyrosine kinases that play an
important role in the development and proliferation of glial tumors.
Key events in their activation are di- (oligo)merization followed by
trans-phosphorylation and downstream signaling cascades. Our aim
was to reveal how cell confluence-dependent PDGFR activation is
regulated by the molecular environment of receptors in the cell
membrane. PDGF receptors were labeled with immunfluorescence. Their
spatial arrangement and relationship to lipid rafts decorated with
fluorescent choleratoxin B subunits (CTX-B) were determined by
confocal laser scanning microscopy. Intracellular calcium levels as a
measure of receptor activation in response to PDGF were measured with
ratio videomicroscopy. The phosphorylation of receptors was assessed
with anti-phosphotyrosine antibody in Western-blot and in situ
immunofluorescence experiments. The glioblastoma cell lines A172
and T98G express primarily PDGFR beta. The number of receptors in
the cell membrane increased as cell cultures reached confluence. Parallel
to this, calcium responses evoked by PDGF were 2-phased and prolonged
in an increasing portion of cells. In contrast, PDGFR mRNA levels
decreased with confluence, while the rate of spontaneous internalization
increased. Receptors showed a non-random, clustered distribution in
the cell membrane. The overlap of receptor clusters with CTX-B-labeled
lipid rafts was substantial. The cross-correlation coefficient characterizing
the overlap increased with cell confluence. Furthermore, receptors
showed higher relative phosphorylation in rafts than outside rafts.
Interestingly, PDGF stimulation increased PDGFR phosphorylation much
more in the non-confluent cells. However, inhibition of tyrosine
phosphatases reverted this situation and the larger extent of spontaneous,
as well as ligand-induced phosphorylation in confluent cells became
apparent. Crosslinking of the lipid rafts by CTX-B at 37 C led to the
aggregation of lipid rafts and sequestration of PDGFR clusters from
them. Reducing the cholesterol content of the cell membrane by methylbeta-cyclodextrin dispersed lipid rafts and PDGFR clusters, decreased
their overlap, and almost completely abolished phosphorylation and
calcium response to PDGF stimulus. We conclude that raft localization
of PDGFR has a functional consequence and is linked to regulation of
proliferation as cells reach confluence inasmuch as both the receptor
and tyrosine phosphatases are increasingly clustered in lipid rafts upon
reaching confluence, whereby a greater turnover of the receptors´
activation cycle allows a more efficient downstream signaling.
43
DEVELOPMENT OF A NEW METHODOLOGY AND
TOOLS FOR PREDICTIVE ANALYSIS OF CYTOMIC
DATA: FINDING COMBINATIONS OF
IMMUNOPHENOTYPIC PARAMETERS WHICH CAN
PREDICT THE OUTCOME OF SEPTIC SHOCK PATIENTS
Jorge Monserrat1, Eduardo Reyes1, Alfredo Prieto1, Angela
Hernández1, Hugo Barcenilla1, Raul De Pablo2, David Diaz1,
Cristina Sánchez1, Guillermo Revilla1, Melchor ÁLvarezMon3
1
Alcala University, Immune system Diseases and Oncology
Laboratory, CNB-CSIC R&D Associated Unit, Alcala de Henares,
Madrid, Spain; 2Hospital Universitario Príncipe de Asturias, Unidad
de Cuidados intensivos, Madrid, Spain; 3University Hospital
“Príncipe de Asturias”, Immune system Diseases and Oncology
Service, Alcala de Henares, Madrid, Spain
Multi organic dysfunction syndrome secondary to septic shock is the
main cause of death in intensive care units (ICU). It is difficult to predict
the risk of death of septic shock patients. Objective: To find a group of
inmunophenotypic variables that could predict the outcome of septic
patients at the moment of admission in the intensive care unit. Materials
y Methods: We have studied peripheral blood lymphocytes from 34
septic shock patients and 36 healthy controls. Peripheral blood sample
extraction was performed at the moment of admission in the ICU.
Immunophenotypic studies of the main lymphocyte populations (CD3,
CD4, CD8, CD19, and CD56) in combinations of tour colors with
activation markers (CD69, HLA-DR, CD25, CD38, CD45RA, CD45RO,
CD71, CD23, CD57), coestimulation (CD28, CD80, CD86, CD40L,
CD40), cell adhesion (CD11a, CD11b, CD11c, CD31, CD62L) and
apoptosis (CD95). Measurements were optimized to maximize the
precision of the cytomic measurements because accurate measurement
of cytomic parameters is essential for its accurate predictive value. Results:
After multiparameter analysis we obtain a data base of 235 phenotypic
quantitative variables. We applied algorithms to convert these
quantitative data into qualitative data (-, 0. +) needed to start the predictive
value analysis. Cut off values were adjusted to obtain the maximum
discrimination power. We also designed another algorithm to establish
a ranking of phenotypic variables based on the differences of their
values between groups of patients with opposite outcomes. Based on
the ROC curves of individual variables we pre-selected the variables
with the highest predictive value. We designed a tool to generate ROC
curves by combining several immunophenotypic variables. We
established the desired levels of sensitivity and specificity for the
prediction of the outcome. Three criteria were considered to select the
combination of variables to include in the prognostic definition: 1 to
include the minimum number of variables, 2 the predictive value of
each variable and 3 the ability to complement the predictive value of
another variables in the combination. We tried several variable
combinations until we find the simplest combination of variables to
predict the out come of the septic shock patients. Conclusion: The
methodology and tools for predictive analysis of cytomic data can find
combinations of immunophenotypic parameters which predict the
outcome of septic shock patients. The methodology developed is useful
to construct combinations of cell parameters with predictive value.
44
STATIC MAGNETIC FIELDS STIMULATE SKELETAL
MUSCLE DIFFERENTIATION
Dario Coletti1, Maria Cristina Albertini2, Sergio Adamo3,
Laura Teodori4
1
University of Rome La Sapienza, Histology and Medical
Embryology, Rome, Italy; 2Institute of Biochemistry G. Fornaini,
Urbino, Italy; 3University of Rome La Sapienza, Rome, Italy;
4
ENEA - Casaccia, Division of Toxicology, Rome, Italy
The development of new strategies aimed to enhance skeletal muscle
differentiation is important in order to obtain large amounts of muscle
in vitro for tissue engineering applications and for therapeutic
applications. Very little is known on the biological effects of static
magnetic fields (SMF) on living cells. To assess the effects of SMF on
differentiating skeletal muscle cells, we cultured rat L6 myoblasts in the
absence or presence of SMF and induced them to differentiate, by
lowering the serum concentration of the culture medium, in the absence
or in the continuous presence of SMF. We noticed a remarkable
hypertrophic effect exerted by SMF on skeletal myotubes. This effect
was observed independently from expression changes and nuclear
translocation of myogenin, a pivotal regulator of myogenesis. Given
the importance of cell-cell interaction and alignment during muscle cell
fusion into multinucleated myotubes, we investigated whether SMF
could affect the cytoskeletal organization in this experimental model.
By phalloidin-FITC labeling we detected SMF-mediated effects on actin
stress fibers consisting in a reorganization of actin filaments observed
in the differentiating myoblasts exposed to SMF. At later differentiation
stages, the actin filament reorganization resulted in a different cell
orientation and cell-cell interaction in cultures exposed to SMF as
compared with the control. In addition, by image analysis at single
myotube level we demonstrated that MF determined a higher actin
content in respect to the control. We have recently reported that
phospholipase D (PLD) plays a crucial role in remodelling the actin
cytoskeleton, a process ultimately affecting skeletal muscle differentiation
in vitro. By incubating the cell cultures in the presence of 0.5% 1butanol, a specific PLD inhibitor, we abolished the SMF effects on actin
accumulation and muscle differentiation, so demonstrating that SMF
effects on muscle cells are PLD-dependent. We observed a robust
differentiation of muscle cells when exposed to SMF even in the presence
of TNF-alpha, a cytokine known to potently inhibit myogenesis in vitro
and in pathological conditions such as cachexia. In conclusion, we here
show that SMF can enhance skeletal muscle differentiation and rescue
differentiation in the presence of TNF-alpha. SMF do not seem to alter
the genetic program activated by low serum levels thus likely triggering
epigenetic mechanisms in muscle cells. We provide the first data showing
that SMF act on PLD function and affect the actin cytoskeleton facilitating
the progress of cell maturation toward the myocyte phenotype. The
evidence that SMF can contrast the cytokine-dependent inhibition of
muscle differentiation represent a hint for therapeutical applications.
45
A SYSTEMATIC APPROACH TO ANALYZING CHANGES
IN PROTEIN SUBCELLULAR LOCATION DURING THE
CELL CYCLE
Elvira Garcia Osuna1, Margaret H. Fuhrman2, Jonathan W.
Jarvik3, Robert F. Murphy4
1
Carnegie Mellon University, Biomedical Engineering, Pittsburgh,
Pennsylvania; 2Carnegie Mellon University, Biological Sciences,
Pittsburgh, Pennsylvania; 3Carnegie Mellon University, Biological
Sciences, Mellon College of Science, Pittsburgh, Pennsylvania;
4
Institute of Technology, Pittsburgh, Pennsylvania
Proteomics is the study of all aspects of protein behavior on a
comprehensive basis. Current work in this field includes systematic
analysis of structure, expression levels, and interactions; and these
projects will provide critical data for understanding and modeling cell
101
and tissue behavior. Knowledge of the subcellular location of each
protein is equally important to this task. Automated methods of
determining protein location patterns from fluorescence microscopy
have therefore been developed and shown to work well for static 2D
and 3D images. However, little work has been done to systematically
describe how location patterns change over the cell cycle. We have
therefore used automated microscopy to construct data sets of 2D timelapse images of 3T3 cell lines expressing different proteins tagged with
Green Fluorescence Protein (GFP). These cell lines were constructed
using CD-tagging as described previously [J.W. Jarvik et al.
BioTechniques 33:852-867]. The cells were also stained with Hoechst
33342 to label DNA so that the cell cycle stage of each cell could be
determined. A third fluorescence channel was used to estimate cellular
autofluorescence, allowing the GFP fluorescence value for each pixel
to be corrected for the contribution of autofluorescence. As an initial
approach to determining whether the distribution of a given protein
changes during the cell cycle, images for each protein were separated
into sets of G1 and G2/M cells and a sensitive hypothesis test was
performed to determine whether the distribution of GFP was statistically
distinguishable between each pair of sets. The basis for this test was the
well-characterized set of Subcellular Location Features (SLF) that we
have previously developed for comparison and classification of protein
patterns [R.F. Murphy, Cytometry 67A:1-3]. The results indicate that,
as expected, a number of proteins undergo significant changes in
distribution as they progress from G1 to G2.
46
EVALUATION OF ANTI-CANCER TARGETS ON
CIRCULATING TUMOR CELLS TO PREDICT
THERAPEUTIC SUCCESS
Arjan Tibbe1, Joost Swennenhuis1, Gerald Doyle2, Dave
Chianese2, Jan Keij2, Chandra Rao2, Mark Connelly2, John
Verrant2, Leon WMM Terstappen2
1
Immunicon Europe Inc., Enschede, Netherlands; 2Immunicon
Corporation, Huntingdon Valley, Pennsylvania
The CellSearch TM System has been used in multi-center prospective
studies to demonstrate that presence of tumor cells in blood of patients
with metastatic carcinomas is associated with poor survival prospects.
Failure to eliminate Circulating Tumor Cells (CTCs) after one cycle of
therapy in these studies strongly suggests that these patients are on a
futile therapy. Assessment of the presence of therapeutic targets on the
tumor should enable the appropriate choice of therapy. Here we
demonstrate that anti-cancer targets can be identified on CTCs before
initiation of therapy. In the CellSearchTM System CTCs from 7.5 mL of
blood are identified as Cytokeratin(CK) +, CD45- nucleated cells after
EpCAM immunomagnetic selection. CTCs are identified by the
CellTracks® Analyzer II at the upper surface of a cartridge where they
are held by a magnetic field. Three of the six colors currently available
in the analyzer are used for the identification of the CTCs and the others
are available for further interrogation of CTCs. Fluorescently labeled
antibodies that recognize treatment targets associated with known
therapies such as HER2, IGF-1, Bcl-2 and EGFR can be assessed on the
CTCs. For expression of therapeutic targets at the molecular level we
demonstrated that CTCs can be preserved for cytogenetic analysis. After
the fluid in the cartridge is removed the cells are fixed and maintain
their original position. Hybridization conditions remove the fluorescent
probes used for the original identification. Since the system knows their
original position, the cells can be reexamined for the presence of probes
of interest. The example shows a CTC and a leukocyte before and after
hybridization (left and right from the dotted line) for chromosome 1, 7,
8 and 17. Grey color in the overlay shows the DAPI stained nucleus
after hybridization. Expression level of specific drug related molecular
targets can be detected on CTCs and can be assessed at the protein or
gene level. In addition, effect of therapy can be assessed after
administration of therapy by a change in the number of the CTC after
days or weeks of therapy rather than months.
102
Calibration and Standardization
47
COMPREHENSIVE CHARACTERIZATION OF FLOW
CYTOMETER FLUORESCENCE MEASUREMENT WITH
A MINIMAL BEAD SET
Robert A. Hoffman1, Joseph Trotter2, Alan Stall3
1
BD Biosciences, San Jose, California; 2BD Biosciences, San
Diego, California; 3BD BioSciences Pharmingen, San Diego,
California
Many instrument performance factors contribute to the accuracy and
precision of flow cytometer measurements of fluorescence. The major
factors are linearity, illumination uniformity, illumination noise, detection
efficiency (Q), optical background (B), and electronic noise. Deviation
from linearity (direct proportionality) is the measure of accuracy. The
combined effect of illumination uniformity and illumination noise
contributes a constant variance to the overall precision (CV) of
fluorescence measurements independent of how bright the fluorescence
signal is. Detection efficiency (the number of photoelectrons generated
per fluorochrome molecule passing through the illumination beam),
optical background and electronic noise are the primary factors that
determine the CV´s of low-level signals. To simply measure all these
fundamental instrument characteristics, we have developed a method of
data acquisition and data analysis using as sample a mixture of beads
with 3 different intensity levels (dim, midlevel and bright). Linearity is
measured using the dual pulse method (ref. 1) by measuring the ratio of
median channel of two bead intensities at varying photomultiplier (PMT)
voltages. Electronic noise is determined from the broadening of a
population CV as the PMT voltage is decreased. The contribution of
illumination variability to measurement variance is estimated from the
bright bead CV. Q and B (ref. 2) are determined from the CV´s of the
midlevel and dim beads respectively after correcting for electronic noise
and illumination variance. Knowledge of the intrinsic bead (sample)
CV is necessary in order to perform an accurate instrument
characterization.
Methods to determine the intrinsic bead CV will be
presented. We will also show examples of instrument characterization
using the 3-bead method and show how the measured characteristics
can be used to predict performance of a wide variety of applications.
REFERENCES 1. Bagwell, C. B., Baker, D., Whetstone, S., Munson,
M., Hitchcox, S., Ault, K. A. and Lovett, E. J. (1989). A simple and
rapid method for determining the linearity of a flow cytometer
amplification system. Cytometry 10, 689-94. 2. Chase, E. S. and
Hoffman, R. A. (1998). Resolution of dimly fluorescent particles: a
practical measure of fluorescence sensitivity. Cytometry 33, 267-79.
48
INITIAL RESULTS FROM NATIONAL SURVEY OF Q
AND B VALUES
Eric Chase1, Raymond Lannigan1
1
Cytek Development, Fremont, California
Initial measurements of Q and b values on several flow cytometers in
the US are presented. The rapid method used to determine Q and b was
discussed in Cytometry (Resolution of Dimly Fluorescent Particles: A
Practical Measure of Fluorescence Sensitivity; E.S. Chase and Robert
Hoffman; Cytometry, 33:267). Duke FC3M beads were used for the
data. Sources of error in the Q and b measurements are presented: log
amp nonconformity, spectral mismatch between the hard dyed beads
and target dyes, filter variation from instrument to instrument, and
MESF assignment of the beads themselves. Measured Q values are
compared to estimates of Q values calculated from the design of flow
cytometers and the dye characteristics. Estimated Q values are within a
factor of 2 of observed values. Ratios of calculated Q values between
FL1 and other channels were used to estimate the accuracy of PE MESF
assignments by various bead manufacturers. Calculated ratios of Q are
expected to be more accurate than the calculated values. Sources of the
large variance of Q from instrument to instrument are discussed.
49
DATA INTEGRITY AND REPEATABILITY IN
CYTOMETRIC MEASUREMENTS
William Ortyn1, David Basiji1, Brian Hall1, Richard Bauer1,
Cathleen Zimmerman1, David Perry1, Keith Frost1, Richard
Esposito1, Thaddeus George1, Philip Morrissey1
1
Amnis Corp., Seattle, Washington
The need for repeatable cytometric measurements from day to day and
between instruments is important for research applications, especially
where conclusions are drawn from the analysis of numerous samples
taken over a long period of time or across multiple instruments. Instrument
variability clouds results leading to hidden costs where false conclusions
are drawn or more testing is required to confirm findings. As analytical
cytometry moves into clinical screening applications, decreasing
instrument variability and ensuring instrument parameters are operating
within designed limits is paramount for decreasing morbidity and
lowering treatment costs through increased sensitivity and specificity of
testing. Ensuring data integrity can be complex as many factors are
involved in the precision of cytometric measurements. This is especially
true for image-based instruments where several hundred features may
be measured on a single cell. The variation of feature measurements is
highly dependent on multiple operating parameters of an instrument
and can exhibit non-linear behavior due to thresholding techniques
employed in segmentation, feature calculations and classification. An
ideal methodology for instrument data integrity testing would include
standardized tests, low cost test samples, parameters with diagnostic
significance, automated recording of results and limit checking and a
comprehensive suite of tests. In this presentation we describe the
Automated Systematic Suite of ImageStream Tests (ASSIST). ASSIST is
a set of comprehensive calibrations, tests and limits employed by the
ImageStream system to ensure the instrument is operating within normal
limits. We describe the operation of ASSIST which is conducted daily
on a low cost engineered bead sample run concurrently with cells during
operation of the instrument. ASSIST is fully automated, requires less
than ten minutes to complete and fully characterizes the performance of
the instrument by measuring, setting and verifying over 2000 parameters.
We describe several tests and calibrations from a suite of tests including
those used to automatically perform two axis excitation laser alignment,
test brightfield illumination uniformity, calibrate pixel gain and offset,
measure spatial alignment of key optical elements, characterize fluidic
core stability, test detector linearity and measure image collection
modulation transfer function. The comprehensive set of tests
incorporated within ASSIST provides the basis for quality control in the
production process, requalifies the instrument after field service actions
and provides an essential step toward assuring data integrity and
repeatability in cytometric measurement.
50
ABSOLUTE FLUORESCENCE CALIBRATION: THEORY
AND PRACTICE
Ian Theodore Young1, Yuval Garini1, Bart J. Vermolen1, Guus
Liqui Lung1
1
Delft University of Technology, Department of Imaging Science &
Technology, Faculty of Applied Sciences, Delft, Netherlands
While fluorescence microscope systems remains an essential tool in
modern biology and medical work, no compact instrumentation has
been developed for the rapid calibration of such systems. Almost
invariably results are presented in terms of the [AU], “arbitrary units”.
To remedy this situation we have developed a small, portable instrument
- the size of a microscope slide - that uses low-power LEDs at different
wavelengths to produce calibrated amounts of light. The instrument through a USB connector - is controlled by a computer so that the
current to the selected LED can be swept through an increasing range of
values. The amount of light measured by the microscope’s total imaging
system (lenses, filters, EO sensor, and digitizer) is then recorded to
provide a “current in, digital value out” calibration. Further, the current
can be translated easily to optical power and thus photons per second at
the chosen LED wavelength. We will 1) present a straightforward
theory of fluorescence production, 2) describe the calibration of the
system that we have built, programmed and tested for accuracy and
precision, and 3) compare it to results for fluorescence calibration beads
with “known” numbers of fluorescent molecules.
LED Microscope Slide and Measurement
51
QUANTITATIVE CHARACTERIZATION OF CONFOCAL
MICROSCOPE PERFORMANCE
Edward H. Cho1, Stephen J. Lockett1
1
SAIC-Frederick, Frederick, Maryland
The confocal microscope is now in wide-spread use for quantitatively
analyzing molecular pathways inside living cells, and therefore
convenient characterization of its performance is essential. However,
methods developed to date for evaluation confocal microscopes are
generally rather time consuming to implement and it is hard to compare
different instruments because they do not yield measurements in absolute
terms. Thus, we built a highly stable, uniform and isotropically-emitting
light source with an intensity equivalent to a dim, fluorescence-labeled
cell sample for calibrating the emission light path of optical microscopes.
The source consisted of a battery-driven light emitting diode placed
behind a near-Lambertian diffuser and housed inside a coverslip
bottomed 35 mm cell culture dish. Since the emission from the source
was both uniform and isotropic, the image intensity did not vary
significantly as a function of the distance from the source to the objective
lens. This had the major practical advantage that the same intensity was
obtained without any precise adjustment when the source was removed
from and replaced on the microscope. We mathematically modeled the
emission light path of laser-scanning confocal microscopes using the
Poission and Gaussian distributions to represent photon loss and
amplification noise respectively. The model enabled us to derive
procedures for measuring the absolute photon detection efficiency,
dynamic range, linearity, uniformity over the detection area,
amplification noise and background noise. Analysis of light source
images acquired using a very common confocal microscope model
showed that 0.3% of photons emitted from a sample are recorded in the
image. This was as high as can be expected given the inherent limitations
of the optical components and photomultiplier tubes (PMT). As
expected, image intensity was proportional to intensity from the light
source and the efficiency of the confocal microscope was independent
of PMT gain. However, interestingly amplification noise was
proportional to gain, leading us to demonstrate that the highest dynamic
range is achieved with relatively low gain and 12-bit digitization. Practical
applications of the light source for checking the transmission of optical
components in the emission light path were tested by exchanging different
components (e.g. objective lens, emission filters, dichroic mirrors) in
the emission light path. Funded by NCI Contract NO1-CO-12400.
52
MEASUREMENT OF STABLITY AND PRECISION OF
LIGHT DETECTION IN OPTICAL MICROSCOPY
Tytus Bernas1, Elikplimi KWAKU Asem2, J. Paul Robinson3,
Bartlomiej Rajwa4
1
Purdue University, Bindley Bioscience Center, West Lafayette,
Indiana; 2Indiana University, Pharmacology, School of Medicine,
West Lafayette, Indiana; 3Purdue University, Basic Medical Sciences
& Biomedical Engineering, West Lafayette, Indiana; 4Purdue
Lafayette, Indiana
Currently available techniques of calibration and standardization of
optical microscopes provide estimation of stability and measurement
precision (noise) of an imaging system at single level of signal. In
addition only the total noise level, but not its characteristics (spectrum),
is measured. We propose a novel technique for estimation of time
variability of signal and noise in microscopic imaging. The method
requires registration of a time series of images of any stationary
microscope specimen. The analysis is multi-step process, which separates
monotonic, periodic and random components of pixel intensity change
in time. The technique allows simultaneous determination of dark,
photonic and multiplicative components of noise. Consequently,
confidence interval (noise) is obtained for each level of signal along
with the respective confidence interval (noise). The proposed algorithm
can be applied to detect mechanical instability of a microscope and
instability of illumination source. In addition, photobleaching kinetics
may be characterized at each level of fluorescence intensity. The
technique is validated using datasets of biological images with known
signal and noise characteristics. The method is then applied to assess
performance of photomultipliers in a confocal microscope and CCD
cameras in a wide-field microscope.
103
Advanced Microscopy and Image Acquisition 2
53
IMPROVING SINGLE MOLECULE FÖRSTER RESONANT
ENERGY TRANSFER MEASUREMENTS BY PULSED
INTERLEAVED EXCITATION AND FLUORESCENCE
CORRELATION SPECTROSCOPY
Steffen Rüttinger1, Benedikt Kraemer2, Martin Roos3,
Eberhard Hildt3, Felix Koberling2, Rainer Macdonald1
1
Physikalisch-Technische Bundesanstalt, Berlin, Germany;
PicoQuant, Berlin, Germany; 3Robert Koch-Institut, Berlin,
Germany
2
Förster Resonant Energy Transfer (FRET) is a well-known effect with
first applications as nanometric spectroscopic ruler dating back to 1978
[1]. With the recent advances in sensitive fluorescence detection
techniques single pair FRET (spFRET) is used to detect e.g. co-localization
of molecules or to measure conformational changes on a single
molecules. However, in spite of the strong distance dependency of the
energy transfer efficiency quantitative results are difficult to obtain and
FRET experiments in solutions are generally interpreted qualitatively.
One problem is the so called zero efficiency peak [2] caused by FRET
pairs with missing or non fluorescent acceptor [3]. Furthermore, the
analysis is hampered by the presence of crosstalk due to imperfect
spectral filtering, direct excitation of the acceptor as well as not directly
measurable excitation and quantum efficiencies of the fluorophores
and sensitivities of both detection channels To identify molecules
diffusing across the interaction volume and exhibiting non-fluorescent
or missing absorbing dye, we applied (dual color) pulsed interleaved
excitation for FRET measurements [4] (PIE-FRET). Combined with
time correlated single photon counting (TCSPC), the presence of a
fluorescing acceptor is detected without relying on the occurrence of
energy transfer. Contributions originating from the zero efficiency peak
can be identified unambiguously and subsequently eliminated to obtain
meaningful FRET-histograms. Since direct acceptor excitation, molecular
brightness of donor and acceptor fluorophores and crosstalk or leakage
is determined by analyzing the same data-set with FCS, all quantities
required to interpret FRET-measurements correctly are available. To
demonstrate the advantages of our approach, we investigated a polyproline assay labeled with Alexa 555 and Alexa 647 as donor and
acceptor, respectively. [1] L. Stryer., Ann. Rev. Biochem, 47:819–846,
1978. [2] A. Deniz et al., Proc. Natl. Acad. Sci. USA Biophysics,
96:3670–3675, 1999. [3] B. Schuler et al., Nature, 419:743–747, 2002;
A. Dietrich et al., Reviews in Molecular Biotechnology, 82:211–231,
2002; S. Weiss, Science, 283:1676-1683, 1999. [4] D. Lamb, Picoquant,
10th International Workshop 2004.
54
EXTENDED DEPTH OF FIELD IMAGING WITH THE
IMAGESTREAM EDF IMAGING FLOW CYTOMETER
SYSTEM
William Ortyn1, David Basiji1, Keith Frost1, Richard Bauer1,
Richard Esposito1, Cathleen Zimmerman1, David Perry1,
Philip Morrissey1, Thaddeus George1, Brian Hall1
1
Confocal microscopy provides the ability to synthesize an image of a
cell from multiple focal planes bringing all features simultaneously into
focus. This capability is desirable for a wide range of cell analysis
applications including co-localization studies, quantifying the
translocation of molecules between cellular compartments and the
enumeration of FISH probes randomly located in a nucleus. In this
presentation we disclose a technique to perform high speed, extended
depth of field (EDF) fluorescence imaging in flow, whereby, imagery
from thousands of cells is collected in less than a minute with the entire
cell simultaneously in focus. We provide a theoretical treatment of the
underlying mechanism and show the discrete steps in the process of
generating EDF imagery. We demonstrate the effectiveness of the
methodology by comparing large focus pans of several thousand beads
collected with the standard ImageStream and the ImageStream EDF
systems. Visual observation of both standard and EDF collection modes
shows that the EDF method maintains good focus while the standard
collection mode exhibits substantial blurring. We then analyze
photometric and morphological bead parameters to quantitatively assess
the benefits of the method. Finally, we apply the method to the
104
enumeration of FISH probes in a comparative study. We show EDF and
non-EDF cell imagery to visually demonstrate the benefits of the method
and then apply an automated classifier to both image sets demonstrating
significant increase in the efficacy of chromosome enumeration using
the EDF imagery.
55
ABOUT A NOVEL LIGHT MICROSCOPE
ARCHITECTURE
Rainer Uhl1
1
Ludwig-Maximilians-Universitat Munchen, BioImaging Center,
Martinsried, Germany
Human Vision comprises a sequence of complex interactions between
eye and brain. The microscope extends this domain into the microcosm.
While conventional light microscopes are a mere extension of the optical
apparatus of the eye, modern imaging microscopes can assume many
more functions usually associated with the retinal part of the eye and the
brain itself. They thus comprise the front-end of a computer-based
intelligence and turn the microscope into a quantitative measuring
device. We will present a novel light microscope architecture (iMIC),
which aims at the highest possible degree of automation and high
throughput. In sharing this goal with other approaches, the iMIC isn´t
restricted to the most fundamental imaging techniques, instead it
constitutes a seamless integration of all conceivable high end imaging
and sample manipulation techniques into a single, highly versatile
instrument. These include: • Time-lapse studies with genuine real-time
performance; • FRET measurements at two freely selectable emission
wavelengths; • TIRF measurements with dynamically variable TIRFangle; • FRAP or other techniques (e.g. laser microdissection, optical
tweezers) requiring the positioning of a laser beam in the object field; •
Widefield, structured illumination or slit-scan confocal measurements
with incoherent illumination at any freely selectable wavelength between
340 and 680 nm. • Multispectral confocal laser-scanning with fully
digital scan-control and close to theoretical sensitivity, and •
Multiphoton excitation experiments with maximal photon collection
efficiency.
56
CELL AND TISSUE RELATED SCANNING
FLUORESCENT VIRTUAL MICROSCOPY USING A
DEDICATED DESK TOP SCANNER SYSTEM
Bela Molnar1, Viktor SEBESTYEN Varga2, Attila
Tagscherer3, Tibor Virag4, Viktor Kamaras4, Zsolt Tulassay2
1
Semmelweis University II, Budapest, Hungary; 2Semmelweis
University, II.Dept. of Medicine, Cell Analysis Lab, Budapest,
Hungary; 3Semmelweis University of Medicine, Budapest,
Hungary; 43DHISTECH LTD, Budapest, Hungary
Background and aims : Scanning fluorescent microscopy on traditional
microscopes are slow, inconvenient and hardly limited in the automation
opportunities. We aimed to develop a desk top size fluorescent slide
scanner for slide loading and identification (upto 10 slides), including
epifluorescent illumination with enhanced filter numbers (8), with
increased fluorescent light source life time and and a modular
construction for application specific camera selection. Methods:In a
public.private partnership (Semmelweis Uni.- 3DHISTECH LTD.,
Budapest, Hungary) a desk top, slide loading and scanner mechanics
was developed incorporating traditional optical elements from a high
quality microscope ( Axioplan 2 Imaging, Carl Zeiss, Germany) without
any ocular. The system performance was tested from the following
point of views: automated slide loading, slide identification, region of
interest determinations, focusing, multichannel slide scanning. The
system´ fluorescent quantitative calibration was done with the X-cyte
fluorescent light source without and with a special bead based algorythm
or using fluorescent slides( FITC, Rhodaminm Hoechst). The linearity
and quantitative accuracy was tested using fluorescent beads. Routine
software applications in cytology and histology were developed towards
pseudocoloured visualisation, counting, TMA in a virtual microscopy
environment. Results: Systems mechanics was robust to load and identify
the barcode labelled slide set (1-10). The systems linearity was proven
in the range of 1 to the 106. The CV of the measured PI labelled
lymphocyte slide was 4.3%, CV of the bead measurements was below
the given exclusion criterias using the fluorescent slide based white
field compensation, only. Multichannel , pseudocolorised cytology
and histology virtual microscopy was easily applied for visual analysis.
Transmitted and fluorescent TMA evaluation was successfully
implemented. Conclusions: Developing a desktop fluorescent slide
scanner and sophisticated virtual microscopy programms the traditional
microscopy based scanning fluorescent microscopy could be enhanced
for everday use in fluorescent cellular and histology research.
57
AFFORDABLE CYTOMETRY FOR INFECTIOUS
DISEASE DIAGNOSIS AND MONITORING
1
2
Howard Shapiro , Nancy Perlmutter
1
2
Howard M. Shapiro, M.D., P.C., West Newton, Massachusetts;
Howard M. Shapiro, M.D., P.C., Allston, Massachusetts
concept for the investigation of non-adherent cells without tethering, at
a single cell resolution. Thus, repetitive, high-content signal and image
analysis of the same non-adherent, non-tethered individual cells, which
are subjected to bio-manipulations (drug or staining procedures), while
maintaining their viability and identity, can be accomplished. This also
allows the correlation between pre and post fixation measurements.
Image Processing and Analysis 2
59
THE SHAPE OF THINGS TO COME: QUANTITATIVE
ANALYSIS OF CELL MORPHOLOGY
Zachary Pincus1, Natalie A Dye2, Kinneret Keren2, Julie A
Theriot2
1
The HIV epidemic now raging in Africa, Southeast Asia, and other
resource-poor areas of the world is accompanied by epidemics of
tuberculosis (TB) and malaria; many patients are simultaneously infected
with more than one of these diseases, and it is thus essential to develop
simple, affordable technology for diagnosis and monitoring of all three.
Flow cytometry has been the “gold standard” method for determining
the CD4+ T cell count in HIV-infected patients; in recent years, simpler
imaging technology has been shown to produce equivalent results.
Although the “gold standard” methods for diagnosis of TB and malaria
are based on direct detection of the infectious agent by transmitted light
or fluorescence microscopy, cytometric approaches to diagnosis and
monitoring of these diseases have largely been rejected as too expensive.
Microscopy-based methods rely heavily on the morphology of
intraerythrocytic parasites in the case of malaria and on the morphology
of bacterial colonies in the case of TB; however, flow cytometry has
shown that the DNA and RNA content of malaria parasites changes
predictably with their morphologic stage, and flow and image cytometry
suggest that the selectivity of fluorescent stains for TB is primarily
dependent on the permeability of the Mycobacterial cell wall to nucleic
acid dyes. Mycobacteria and malaria parasites stained with appropriate
dyes can readily be detected by a small, simple, inexpensive lowresolution image cytometer incorporating high-intensity light-emitting
diodes (LEDs) as light sources for fluorescence excitation, camera lenses
or low-power microscope optics for light collection, and a chargecoupled device (CCD) or metal oxide semiconductor (CMOS) camera
for detection. This could provide more rapid and precise diagnosis of
disease, determination of drug susceptibility, and assessment of therapeutic
effects than is now possible even in affluent countries. A similar
instrument should also be usable for CD4+ T cell counting and a variety
of other applications in immunology and microbiology. Portions of
this work were supported by NIH Grants AI060272, AI063833, and
HL080898.
58
ENABLING REPETITIVE PROLONGED
MEASUREMENTS OF NON-TETHERED NONADHERENT INDIVIDUAL CELLS IN A MICROTITER
PLATE
Mordechai Deutsch1, Naomi Zurgil1, Elena Afrimzon1, Yana
Shafran1, Assaf Deutsch1
1
Biophysical Interdisciplinary Schottenstein Center for Research and
Technology of the Cellome, Physics, Bar Ilan University, RamatGan, Israel
The use of non-adherent cells, primary cells or cell lines, in cell-based
assays (whether in low- or high-throughput systems, or high content
screening), is slow to appear, despite their tremendous importance in
drug discovery and cell therapy. In order to monitor complex cellular
responses to a variety of biological modulators, it is crucial to be able to
trace temporal behavior of cells at a single cell resolution throughout
various segments of time. While such requirements can be achieved
using adherent cells grown on microtiter plates, it is impossible with
non-adherent cells, without tethering. In the novel approach presented
here, each of the wells within a multi-microtiter plate (96 wells or other)
is fully padded with highly dense continuous array of addressable micro
concave lenses, serving as PicoWells (PWs). A typical well of the 96well plate contains up to 70,000 PWs - each PW is designed to
accommodate a single non-adherent living cell. Fluid, drug and reagent
exchange in the wells is enabled, while keeping the individual cells at
their original acquired locations. The approach presented in the current
study enables, for the first time, the adaptation of the microtiter plate
Stanford, California; 2Stanford University, Biochemistry, School of
Quantitative descriptions of cell shape are necessary for statistical analysis
of morphological variability. While it is possible to define ad hoc metrics
to quantify specific shape phenotypes, tools to intuitively describe and
summarize trends in population morphology do not exist. We therefore
sought a general method to describe cell shapes with parameters that are
biologically meaningful, able to capture any form of shape variation,
and not specified a priori in a potentially biased manner. From a given
set of cells, we create a “principal component shape model” by
performing principal components analysis on a signed distance map
representation of the cell shapes. Such a shape model contains information
about the average shape, the major modes of shape variance in a
population (e.g. large vs. small, round vs. elongated, etc.), and the
relative importance of those modes. Experimental evidence indicates
that modes of shape variation so derived closely mirror qualitative
descriptions of cell morphology used by biologists to describe a given
cell type; thus these modes are quite intuitive. With such a model, the
morphology of individual cells can be quantified, sets of images
representative of a population can be produced, morphological variability
between cell populations can be intuitively visualized and statistically
compared. First, given a particular shape model, a cell shape can be
decomposed into the relative contributions from each mode of shape
variation. This transforms a shape into a small set of numerical parameters
that describe the cell along intuitive axes that are, by construction,
maximally descriptive for a given data set. Moreover, these axes can
be visualized by using the shape model to synthesize shapes that describe
the variation encoded by each shape mode. This visualization provides
a useful and compact summary of the major morphological variation in
a given set of cells, and provides a better summary of a large data set
than a few “representative images”. Finally, quantifying cell morphology
in an unbiased fashion allows us to answer open-ended questions such
as “are cells of population X morphologically distinct from cells of
population Y?” Cells can be plotted according to their shape parameters
to visualize an entire data set and see general trends in cell shape across
populations; such distributions of cell shape can also be statistically
compared to measure differences between cell populations. These
methods have allowed us to quantify, visualize, and statistically validate
the effects of drug treatment on cell shape in Caulobacter and in fish
epithelial keratocytes. Additionally, we have applied this analysis to
movies of crawling keratocytes to understand dynamic shape-changing
during cell crawling.
60
BUILDING GENERATIVE MODELS OF SUBCELLULAR
LOCATION PATTERNS
Ting Zhao1, Robert F. Murphy1
1
Accurate knowledge of the subcellular locations of all proteins will be
necessary for a thorough understanding of cell behavior, both under
normal conditions and in disease. Previous work has demonstrated that
automated classifiers can be used to determine subcellular locations
from fluorescence microscope images with high accuracy. However,
assigning proteins to locations is not sufficient to simulate cell behavior
because it does not provide models of the identified spatial distributions.
We have therefore developed approaches to build object-based generative
models of location patterns. We started by using cluster analysis to
identify the types of possible subcellular objects in a large collection of
105
cell images. Each image of a given pattern is then represented as a set
of objects labeled with types, and the overall pattern is modeled by the
statistical distribution of the number of objects of each type and the
distributions of distances between the nucleus and the objects for each
type. As an initial approach to converting this statistical model to a
generative model, we formed images of a given pattern by randomly
picking objects from real images according to their types and placing
them at positions generated from the distance distribution. We have
used this method to generate images of all major location patterns in
HeLa cells. As a further advance, we built models that can generate the
objects themselves. We synthesized objects in two steps, first generating
shapes and then textures. The parameters necessary for these steps were
learned from the image collection for a given pattern. The quality of
the generated images was assessed by determining whether they could
be recognized by an automated classifier built from the same collection.
The work provides an important new capability for studies of protein
location patterns.
61
IMAGE CYTOMETRY PROFILING OF CELL CYCLE
PHENOTYPES IN GENOME-WIDE SIRNA KNOCKDOWN
CELLS
Yan Feng1, Jonathan Hoyt2, Yong-Chuan Tao2, Timothy
Mitchison3
1
Novartis Institute for Biomedical Research, Genome and Proteome
Sciences, Cambridge, Massachusetts; 2Cambridge, Massachusetts;
3
Harvard Medical School, Systems Biology, Boston, Massachusetts
We used a multi-parameter imaging cytometry based assay and genomewide siRNA knockdown to characterize genes and pathways that regulate
cell cycle progression. A series of statistical methods, including supervised
and unsupervised classification, and genome-wide screen normalization,
were used to identify cell cycle stage of each individual cell and generate
a profile for each gene knockdown. A series of tests were used to
identify potential false-negative or off-target effects. System level
analysis was then performed by putting the cell cycle profile of each
gene knockdown into functional genomics context. Implication of new
cancer drug targets will also be discussed.
62
IMAGE ANALYSIS FOR STRUCTURAL GENOMICS
REVEALS NOVEL PROCESSES IN TUMOR
PROGRESSION AND APOPTOSIS
Bart J. Vermolen1, Ian Theodore Young1, Sabine Mai2, Sherif
Louis2, Vered Raz3, Yuval Garini1
1
Delft University of Technology, Delft, Netherlands; 2University of
Manitoba, Winnipeg, Manitoba, Canada; 3Leiden, Zuid-Holland,
Netherlands
Major success has been achieved in recent years in understanding the
function of the genome. It also became evident that the structure and
organization of the genome in the nucleus is important, it changes
along the cell cycle and during cancer progression. For structural and
organizational studies to take place, it is required to apply markers,
probe regions of interest (wetware), and acquire images (hardware),
tasks that have reached a mature state. For successful interpretation, a
last step has to be made: analysis of the images (software). To obtain
quantitative results, the first two components (wetware and hardware)
have to be taken into account in the software and algorithms. We
studied the spatial organization of telomeres and centromeres. Telomeres
were studied during the cell cycle of normal mammalian cells and after
c-Myc deregulation and centromeres were studied in normal, senescent
and apoptotic human mesenchymal stem cells. We will present the
algorithms that were developed and some of the results. Novel processes
have been discovered, including the cell-cycle dependence of the
telomeres, telomere aggregates during tumor progression and the redistribution of centromeres during apoptosis.
106
63
MICRONUCLEI FORMATION, MORE THAN MEETS THE
EYE? A MULTIPARAMETRIC HIGH CONTENT
ANALYSIS STUDY USING LASER SCANNING
CYTOMETRY
Raffi Manoukian1, Satin Sawant1, Gloria Juan1
1
Amgen, Inc., Thousand Oaks, California
Genotoxicity leading to chromosomal damage during cell division is an
important consideration when screening for novel small molecule
compounds in vitro or for monitoring their effects in vivo. Chromosomal
mutation not only leads to a genotoxic and cytotoxic state but also may
play an important role in carcinogenesis. Under such conditions,
chromosome fragments or lagging whole chromosomes accumulate in
the cytoplasm in anaphase, as they are unable to reach the spindle poles
during mitosis. These residual minute chromatin masses, which are
adjacent to the larger main nucleus, are called micronuclei (MN). The
presence of micronuclei in cells can thus be used as a means to quantitate
chromosome damage. Recent work has shown that High Content Analysis
(HCA) MN assays allow for a more automated, objective, and faster
approach, and yet these and other studies were limited to a single cell
line. Other groups have shown that using one cell line as a standard for
MN could be misleading, as it seems that onset and severity of
micronucleation vary greatly between lineages. In this study we examine
multiple and more clinically relevant cell lines as well as white blood
cells from the peripheral blood in an attempt to not only enumerate MN
formation but to correlate it to multiple endpoints such as cell cycle
modulation, DNA synthesis inhibition and apoptosis. This
multiparametric approach can be achieved by performing high content
analysis using laser scanning cytometry and quantitative imaging as the
investigative platform. We demonstrate that HCA can extract more indepht information, resulting in a correlative matrix of MN formation,
cell lineages and functional endpoints.
64
HISTONE HYPERACETYLATION CAUSES
REPOSITIONING OF CENTROMERES AND
TOPOLOGICAL REORGANISATION OF CHROMATIN IN
HUMAN PROSTATE CANCER
Vicky Kyle2, Perry Maxwell3, Peter Hamilton3
1
Queen’s University Belfast, Centre for Cancer Research and Cell
Biology, Biomedical Imaging and Informatics, Belfast, Northern
Ireland, United Kingdom; 2Napier University, Edinburgh,
Biomedical Science, Edinburgh, Scotland, United Kingdom;
3
Queen’s University Belfast, Pathology, Belfast, Northern Ireland,
United Kingdom
Quantitative changes in nuclear chromatin and epigenetic status occur
in prostatic neoplastic progression, suggesting a functional role for
nuclear topology in tumour development. In vitro studies on normal
and malignant prostate cell lines highlighted quantitative differences in
the chromatin organisation of G0 phase nuclei using high resolution
texture analysis. The basis for changes in chromatin phenotype in prostate
cancer are unknown, however, we expect that histone acetylation may
play a central role. The aim of this study was to explore the impact of
Trichostatin A (TSA) induced histone hyperacetlyation on centromeric
topology and chromatin organisation in prostate cancer cells. Prostate
cancer cell lines LNCaP, DU145 and PNT1A were grown onto glass
slides and treated with 0, 12 and 100 ng/ml TSA for 24 hours. High
resolution computerised texture analysis was used to measure changes
in chromatin phenotype. Centromeric FISH was carried out for
chromosome 11 using a biotin labelled probe, labelled with an FITC
anti biotin secondary antibody and the nuclei counterstained with
propidium iodide. 2D measurements of intranuclear centromere position
were made using Leica QWin. 3D Confocal microscopy was used to
obtain serial optical sections of nuclei approximately 0.12µm apart.
Serial sections were reconstructed using Analyze software
(AnalyzeDirect) to reconstruct and render 3D composite images and a
variety of measurements were made. It was shown that induced histone
hyperacetylation by low dose TSA resulted in increased chromatin
density and chromatin reorganisation. Untreated LNCaP nuclei show
distinct centromeric topologies using 3-D FISH. The PNT1A and DU145
cell lines were most similar where the volume for centromeres was
similar within and between nuclei. In trisomic nuclei, two of the
centromeres were the same volume and one was approximately twice
that size. The largest centromere was positioned equidistant from the
two smaller sized centromeres and was closer to the centre of the nucleus.
2-D image analysis showed that increased chromatin density, was
associated with concomitant rearrangement of chromosome 11
centromeres. In general, the centromeres separated, one moving to a
central position while the other moved to a more peripheral location.
Interestingly, when a third centromere was present, this did not impact
the distance between the smaller centromeres, nor did its position change.
The study of nuclear topology provides important insights into tumour
development and the impact of epigenetic modifiers such as TSA on
nuclear phenotype. In prostate cancer, cells show a unique centromeric
arrangement for chromosome 11 which is modified following low doses
of TSA which alter the global histone acetylation status of the genome.
65
LIGHT EMITTING DIODES (LEDS) AND RED DIODE
LASERS FOR LOW COST MULTICOLOR FLOW
CYTOMETRY
Robert A. Hoffman1, David W. Houck1
1
BD Biosciences, San Jose, California
Multicolor flow cytometry is routinely done using UV, violet, blue and
red lasers. With the exception of red lasers, the cost of these light
sources is thousands of dollars each. LEDs are a potential alternative to
lasers for the entire spectrum of UV and visible light sources. The main
limitation of LEDs is the intensity of the illumination, which is limited
by the brightness of the emitter (power per unit area) and cannot be
increased by imaging the LED emission to a small spot. Compared to a
laser focused to a spot size of 20ìm by 60 ìm, an LED with the same
optical power is one hundredth as intense. The low LED intensity can
be partially compensated for by taking advantage of the extended source
and optimizing the optical and flow conditions. By increasing the time
a particle spends in the excitation beam from 4 ìs to 40 ìs, a ten-fold
increase in signal is obtained. With high power LEDs currently available,
this provides integrated fluorescence signals equivalent to those generated
by lasers with power in the 1- 10 mW range. LED wavelengths
commonly available include 365, 405, 455, 470, 505, 530, 590, and
624 nm. For simplicity of light scatter measurements and high excitation
intensity, a red diode laser with emission in the 635- 640 nm range is a
good alternative at modest cost compared to a red LED. An additional
feature of LEDs or red diode laser is the ability to be rapidly turned on
and off, creating the possibility of flashing various excitation wavelengths
at a particle as it passes a sensing zone in flow. Examples of 4 or more
color analyses will be shown. The multicolor analyses include various
combinations of immunofluorescence, DNA content, viability, side
population for stem cells, and Indo-1 calcium response. Where particle
event rates of a few thousand cells per second are adequate, LED
excitation offers a wide range of application possibilities and flexibility
at relatively low cost.
66
LOW COST LIGHT SOURCE & MINIATURE DETECTORS
YIELD HIGH PERFORMANCE IN A SLOW-FLOW
SYSTEM
light source (532 nm), inexpensive miniature Hamamatsu PMTs, and
minimal analog electronics (prior to and within the DiDAC system) we
demonstrate > 4 full decades of dynamic range on 24-bit area data, with
sensitivity to < 70 MEPE, and with all 8 peaks baseline resolved in
several emission wavelength ranges. Furthermore, 90 degree light scatter
clearly distinguishes 1.87, 2.1 and 2.8 micron diameter polystyrene
microspheres. Only low voltages are required for the pre-amps and
PMTs with the total power used (outside of DiDAC) at < 2 W. This work
was supported by NIH RR020064-01 and NIH RR001315-23
67
LOW COST HAND PORTABLE FLOW CYTOMETRY
Steven W. Graves1, Gregory Kaduchak1, Gregory R.
Goddard1, Robert C. Habbersett1, Michael D Ward1, John C.
Martin1, Mark Naivar1
1
Los Alamos National Laboratory, National Flow Cytometry
Resource, Los Alamos, New Mexico
The development of inexpensive highly portable flow cytometers will
provide powerful solutions for medical diagnostics in third world
countries and point of care testing in physicians offices as well as supply
an analytical platform for homeland defense first responders. The
creation of a truly portable low cost flow cytometer will require a
holistic approach that addresses the primary drivers of cost and portability:
the requirement for large volumes of sheath, a high intensity light
source, and a high speed multiparameter data system. To develop such
a system, we first addressed the requirement of sheath for hydrodynamic
focusing by constructing a sheathless flow cytometer that uses acoustic
energy to focus particles to the center of the flow cell without the
concurrent acceleration that results from hydrodynamic focusing. This
allows us to maintain the advantages of hydrodynamic focusing (e.g.
precise analysis, resolution of free vs. bound probe) and offers additional
key advantages such as high particle analysis rates at extended transit
times as a result of the concentration of sample to the center of the flow
stream. We will present data from the acoustically focused flow cytometer
that demonstrates analysis of hundreds of microspheres per second with
fluorescence CVs of approximately 5% with sensitivity and resolution
approaching that of a conventional flow cytometer. Second, to
demonstrate the advantages of extended transit times, we have used a
miniature low cost laser source that uses approximately 0.5 watts of
electrical power on a slow flow flow cytometer equipped with low cost
miniature PMT modules. We will present data obtained from analysis of
commercial fluorescent microspheres that demonstrate that this system
can obtain fluorescence CVs of less than two percent and detect a few
hundred fluorophores per particle with an optical train (detectors
included) that costs approximately one thousand dollars. Third, the
above systems have been married to a portable digital data acquisition
platform (DiDac II) that has a simple path forward to a low cost miniature
data acquisition system. Finally, we will present data on a prototype
instrument that combines sheathless acoustic focusing with miniature
low power light sources and miniature detectors critical steps in the
development of a battery operated hand-held sheathless flow cytometer
that will cost less than $5000, while providing the sensitivity, precision,
resolution and particle analysis rates commonly associated with modern
commercial benchtop flow cytometers. This work was supported by
NIH RR020064-01, NIH RR001315-23 and DOE LDRD funding.
Robert Habbersett1, Jimmy Parson1, Steven Graves2
1
Resource, Los Alamos, New Mexico; 2Los Alamos National
Laboratory, Bioscience, Los Alamos, New Mexico
We have evaluated a range of miniature, low-cost, low-power, easy to
use detectors and light sources, and – quite frankly – were astounded by
their performance in a simple slow-flow system (developed primarily
for DNA fragment sizing), which has been embellished with forward
and high-angle light scatter detectors, in addition to two fluorescence
channels. In general, reducing the cost, complexity, and size of an
analytical system is desirable, as long as performance is not seriously
compromised. To build a low-cost truly portable instrument, these
considerations become paramount. A central factor in this highsensitivity system is the transit time, which was ~ 200 microseconds
(limiting the throughput to < 5000 events/sec). Using our new digital
data acquisition system (DiDAC-2), we present results - based primarily
on Spherotech RCP-30-5A beads - to demonstrate resolution, sensitivity
and dynamic range on par or better than state-of-the-art commercial
cytometers. With excitation energy from a low-cost monochromatic
68
FAST AND PARALLEL MICROFLUIDIC OPTICAL
SORTERS ENABLE SAFE AND QUICK PURIFICATIONS
OF RARE CELLS
Ruud Hulspas1, Manish Deshpande1, John Gilbert1
1
Cytonome, Boston, Massachusetts
An increased demand for obtaining large numbers of rare cells from
human cell samples is driving flow sorter technologies towards new
frontiers. Microfluidic particle switch based sorting does not require a
droplet or aerosol phase, and can thus be designed to take place in fully
enclosed systems. Hence, microfluidic sorting is the technology of
choice when BSL-4, -3 and even BSL-2 conditions are required and
when cells of interest are best identified by means of flow cytometry.
Due to displacement of relatively large liquid volumes, reliable
microfluidic switch sorting has been slow compared to droplet sorters.
Recently, microfluidic switch technology has been developed to
minimize the disruption in velocity due to fluid displacement.
107
Consequently, we have demonstrated single microfluidic switch sorting
rates at 2000 sorts per second. Overall sample throughput and sort rates
can be further increased through parallel implementation of this
technology in which 10 to 100 microfluidic sorters are fabricated on a
single chip. This paper discusses reliable sorting of human rare cell
populations using a scalable, closed, high-throughput optical sorting
system.
69
DESIGN OF A HIGH-THROUGHPUT BIOMEMS
MICROFLUIDIC CYTOMETER/SORTER
James F. Leary1, Rashid Bashir2
1
Engineering, West Lafayette, Indiana; 2Purdue University, Electrical/
Computer Engineering & Biomedical Engineering, Engineering,
West Lafayette, Indiana
BioMEMS (Bio MicroElectroMechanical Systems) microfabrication
technology is being designed, constructed, and tested for applications
in genomics, proteomics, and drug delivery. A current limitation of
BioMEMS microfluidic technology is overall cell throughput rates. A
multidisciplinary team from our two laboratories is designing a novel,
small, portable, high-throughput (> 100,000 cells/sec) exponentially
staging system that can be used for these and other applications.
The
overall preliminary design (US patent filed and pending) consists of a
disposable microfluidic chip of branching tree architecture for parallel
and multi-stage processing. The system does not need to handle single
cells in its initial fluidic stages. However, by the third or fourth fluidic
stage, single cells can be sorted depending on the overall throughput
rates and initial cell concentration. It is a closed system, thus eliminating
biohazardous aerosols, and also facilitating sterility.
The fluidic
portion of the system (PDA-sized) can be placed, if desired, in a biohazard
hood. The microfluidic chip is made of optically semi-transparent PDMS
((poly)dimethylsiloxane)), with built-in microfabricated, elastomeric
valves. The excitation sources are small, inexpensive, super-luminescent
LEDs (light emitting diodes) requiring only battery-level power.
Photodetectors consist of avalanche photodiodes (APDs). The LEDs
and APDs are arranged in patterns corresponding to the physical structure
of the removable PDMS microfluidic chip which is placed between
them. The present data acquisition system uses a National Instruments
PXI data acquisition system with embedded controller running RealTime LabView version 7.1 and 16-bit multifunction DAQ modules for
digital signal processing and sort logic control.
Preliminary data
from this BioMEMS system will be presented as well as a discussion of
the problems and promises of these new microfabricated approaches to
flow cytometry and cell sorting.
70
A HIGHLY AUTOMATED PARALLEL SORTING (HAPS)
SYSTEM THAT PROCESSES 2.5 X 109 CELLS PER HOUR
UNDER THE CONTROL OF A SINGLE OPERATOR
Gary Durack1, Paul Weiss1
1
iCyt Visionary Bioscience, Champaign, Illinois
Highly Automated Parallel Sorting (HAPS) can now be accomplished
using a new type of cell sorting system that can process many times
what is possible with a traditional FACS device. We demonstrated a
HAPS configuration that can process 2.5 x 109 cells per hour. The
system consisted of 16 HAPS channels, each operated with a throughput
of approximately 50,000 cells per second. Each HAPS channel generated
droplets at 80 KHz yielding a total system droplet output of 1.26 MHz.
The HAPS system was controlled by a single operator through a graphical
user interface (GUI). The HAPS system was used to measure, classify
and sort 1.2 x 1010 cells over a four hour period. Recovery, purity and
efficiency of HAPS were compared to traditional FACS which could
classify and sort 4 x 108 cells over the same period. The HAPS system
is scalable and can be expanded to achieve throughputs of several
millions of cells per second.
108
Cell Physiology 2
71
FLOW CYTOMETRIC MEASUREMENTS OF OXIDATIVE
STRESS IN HEMOGLOBINOPATHIES
Fibach Eitan1, Johnny Amer2
1
Hadassah University Hospital, Jerusalem, , Israel; 2Hadassah
University Hospital Jerusalem, Hematology, Jerusalem, , Israel
Oxidative stress represents an imbalance between oxidants and
antioxidants. Oxidants include Reactive Oxygen Species (ROS), unstable
reactive free-radicals possessing an unpaired electron, which can oxidize
various molecules leading to cell damage. They are produced
continuously in cells as by-product of metabolism and can be increased
by environmental factors and pathological conditions. The antioxidants
include reduced glutathione (GSH) - the most potent cellular ROS
scavenger. Hemoglobinopathies are inherited disorders which result
from a decreased synthesis of hemoglobin in thalassemia, or synthesis
of an abnormal hemoglobin in sickle cell anemia (SCA). Although the
primary lesion is in the globin genes, the clinical symptoms of these
diseases could be mediated by oxidative stress. We developed flow
cytometric techniques to measure multiple aspects of oxidative stress in
various blood cells. ROS generation was measured by staining the cells
with 2´-7-dichlorofluorescein; GSH – by staining with mercury orange;
membrane lipid peroxidation – with fluor-DHPE and externalization of
phosphatidyl serine (PS) moieties, a marker of membrane damage, by
fluorochrome-conjugated Annexin-V. Specific sub-populations were
gated based on their forward and side light scatter as well as by staining
with antibodies to lineage-specific surface antigens, glycophorin A for
RBC, CD61 for platelets and CD15 for neutrophils. Thus, various
oxidative stress parameters could be assigned to each cell type. Our data
indicate that cells derived from patients with beta-thalassemia or SCA
have higher levels of ROS, PS and lipid peroxidation and lower levels
of GSH compared with their normal counterparts. The results suggest
that hemolysis, the major clinical feature, as well as thromboembolic
complications and recurrent infections, which frequently occur in these
diseases, could be the result of oxidative stress in the RBC, platelets and
neutrophils, respectively, of these patients.
Antioxidants, such as Nacetyl cysteine, vitamin C and vitamin E, reduced the oxidative stress of
these cells and protect them from its deleterious consequences. This was
observed by treating blood cells in vitro as well as in vivo – in a mouse
model of beta-thalassemia and in patients treated with antioxidants.
The results suggest that flow cytometry, a standard technology in most
hematological labs, can be useful for measuring the oxidative status of
various blood cells and for studying the effects of antioxidants or ironchelators in patients with thalassemia and SCA as well as in other diseases
where oxidative stress is involved in their clinical symptoms.
72
ESSENTIAL REQUIREMENT OF REDUCED
GLUTATHIONE FOR THE ANTI-OXIDANT EFFECT OF
THE FLAVONOID QUERCETIN
Roberta Ferraresi1, Erika Roat1, Leonarda Troiano1, Enrico
Lugli1, Chiara Giovenzana1, Maria Garcia Fernandez2, Elisa
Nemes1, Milena Nasi1, Marcello Pinti1, Andrea Cossarizza1
1
Sciences, Modena, Italy; 2University of Malaga, Dept. of
Physiology, Malaga, Spain
Quercetin (3,3´,4´,5,7-pentahydroxyflavone, QU) is one of the most
abundant dietary and frequently studied flavonoids, molecules with a
variety of effects, including those patho-preventive, anti-oxidant, antiallergenic, anti-inflammatory, anti-proliferative and anti-viral. Qu shows
an apparent opposite double action: in different models, it acts at the
same time as a pro- and anti-oxidant. The mechanisms of these effects
have not yet been completely clarified. We have studied by polychromatic
functional flow cytometry the anti- or pro-oxidant effects of Qu. The
U937 human cell line was treated with Qu 10, 50 and 100 uM for
different periods of time, up to 24 hours. In living cells, we evaluated
simultaneously several parameters, including hydrogen peroxide content
by 2,7-dicholorodihydrofluorescein diacetate, superoxide anion content
by hydroethidine, reduced glutathione (GSH) content by
monobromobimane, mitochondrial membrane potential (MMP) by JC1, DNA content by Hoechst 33342, early/late apoptosis by
phosphatidylserine exposure on the outer face of the plasma membrane
by Annexin-V Alexa Fluor 647 and cell viability by Propidium Iodide.
A 16-parameters CyFlow ML (Partec GmbH, Muenster, Germany),
equipped with a blue solid state laser (488 nm, 200 mW), a UV Mercury
lamp HBO (100 long life, 100 W), a red diode laser (635 nm, 25 mW),
a green solid state laser (532 nm, 50 mW) and a CCD camera was used.
For short periods of treatment, Qu exerted an anti-oxidant effect
(decrease in hydrogen peroxide levels), whereas for long periods it
showed a pro-oxidant activity (increase in superoxide anion),
simultaneous loss in GSH content, depolarization of MMP and apoptosis.
Thus, it appears that prolonged/strong treatments with Qu induce
depletion of intracellular GSH stores, so that oxygen free radicals are no
longer scavenged and the pro-oxidant effect prevails on the anti-oxidant
effect. In conclusion, Qu exhibits a double action, anti- and pro-oxidant,
which seems to depend on the cell oxidative balance and GSH content.
Probably, when anti-oxidant system becomes inefficient, the consequent
overproduction of oxygen radicals alters the cell redox state, activating
the apoptotic program. The polychromatic flow cytometric approach,
which has been used to obtain these data, is likely the best technology
for understanding and better clarifying at the single cell level the complex
relationship among anti-proliferative, pro-apoptotic, anti- and prooxidant effect of Qu as well as of other molecules.
73
MITOCHONDIRAL COMPLEX I INHIBITOR ROTENONE
INDUCES CELL DEATH BY ENHANCING REACTIVE
OXYGEN SPECIES GENERATION AND
PEROXYNITRITE-INDUCED CYTOTOXICITY IN HL-60
CELL
Jia Liu1, J. Paul Robinson2
1
Purdue University, Basic Medical Sciences, Veterinary Medicine,
& Biomedical Engineering, West Lafayette, Indiana
Rotenone is able to induce mitochondrial complex I substrate-supported
reactive oxygen species (ROS) production both in isolated mitochondria
as well as in a variety of cultured cells. It also has been suggested that
mitochondria may be sources of nitric oxide (NO) and the primary
targets of NO in the cell. Even a small amount of NO in the mitochondrial
matrix can inhibit mitochondrial respiration. Peroxynitrite is formed
when superoxide and nitric oxide are produced at near equimolar ratio.
As superoxide level rises, it is likely that peroxynitrite generation also
increases in mitochondria. Despite its non-radical nature, peroxynitrite
which is highly reactive, can initiate lipid peroxidation, cause DNA
breakage and induce protein modifications including protein oxidation
and nitration. Both reactive oxygen species and peroxynitrite play an
important role in apoptosis. In addition, peroxynitrite-induced
overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) can
consume NAD+ and consequently lead to ATP depletion culminating in
necrosis. Apoptosis and necrosis could share common initiation
pathways, and cellular ATP levels determine the mode of cell death
(apoptosis versus necrosis). This presentation will show the cytotoxic
effects (apoptosis and necrosis) of superoxide and peroxynitrite induced
by inhibition of mitochondrial complex I by rotenone in HL-60 cell.
Regulatory mechanisms such as antioxidant status, nuclear factor kB
(NFkB) activation, protein phosphorylation will also be presented.
74
A COMPLEX DIETARY SUPPLEMENT DRAMATICALLY
ABERRATIONS, 8-OHDG LEVELS AND H2AX FOCI IN
MICE EXPRESSING ELEVATED FREE RADICAL
PROCESSES
Jennifer A. Lemon1, C. David Rollo2, Douglas R. Boreham1
1
McMaster University, Medical Physics and Applied Radiation
Sciences, Science, Hamilton, Ontario, Canada; 2McMaster
University, Biology, Science, Hamilton, Ontario, Canada
The repair of double-strand breaks (DSBs) is critical for the maintenance
of genomic integrity. Chromosome aberrations (CAs)are most often the
byproduct of unrepaired or misrepaired DSBs, and have been linked to
higher risk of carcinogenesis, abnormal cell function and increased
sensitivity to endogenous metabolic free radical production and DNA
damaging agents. Mice expressing elevated endogenous free radical
processes (TGM) are considerably more radiosensitive than normal
mice as indicated by significantly increased number of CAs when exposed
to a 2Gy in vivo whole body dose of gamma radiation. A complex
dietary supplement designed to offset oxidative stress and associated
cellular processes (i.e. inflammation, mitochondrial dysfunction and
membrane deterioration) dramatically reduces radiation-induced CAs
in both TGM and normal mice. We postulated that one of the main
processes associated with the reduction in radiation-induced CAs involved
increased scavenging of free radicals generated by the ionizing gamma
radiation. To determine if this mechanism played a significant role in
reducing DNA damage and the number of DSBs, we examined the level
of 8-OHdG and the number of ãH2AX foci generated in diet supplemented
and unsupplemented TGM and normal mice exposed in vivo to 2Gy
gamma radiation. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of
the most abundant oxidatively modified lesions in DNA. The levels of
8-OHdG are known to increase with chronic oxidative stress as well as
exposure to ionizing radiation, as a consequence it is frequently used as
a biomarker of DNA damage due to oxidative stress. Preliminary results
indicate that untreated TGM have higher constitutive and radiationinduced levels of 8-OHdG compared to normal mice.H2AX is activated
at the site of DSBs as part of the repair complex and rapidly inactivates
when repair is complete. Although its exact function is still under dispute,
these properties make ãH2AX an ideal marker for determining both the
amount of DNA damage (number of foci) and the rate of DNA repair
(rate of foci disappearance). Untreated TGM and normals respond to
irradiation with similar levels of ãH2AX, suggesting that, although TGM
and normal mice have similar numbers of constitutive and radiationinduced DSBs, a greater proportion of DSBs are misrepaired in TGM
compared to normal mice. We are currently examining 8-OHdG and
ãH2AX levels in supplemented mice, preliminary results show a
significant reduction in both 8-OHdG and ãH2AX, which may indicate
the protective effect of the dietary supplement comes from scavenging
free radicals and preventing DSBs. We will present data on the effects of
the dietary supplement on CAs, 8-OHdG and ãH2AX levels in the bone
marrow of TGM and normal mice.
75
STATIC MAGNETIC FIELDS MODULATE CHEMICAL/
PHYSICAL INDUCED APOPTOSIS AND DNA DAMAGE
BUT NOT OXIDATIVE STRESS IN GLIOBLASTOMA
PRIMARY CELLS
Claudio Panzarella1, Maria Giovanna Valente1, Gianluca
Vigiliano1, Donatella Tirindelli1, Maria Cristina Albertini2,
Luigi Campanella3, Mario Barteri3, Cecilia Costanza3,
Natale Santucci4, Laura Teodori1
1
ENEA CR CASACCIA, BIOTEC-MED, Rome, Italy; 2Institut of
Biochemistry G. Fornaini, University of Urbino, Urbino, Italy;
3
University of Rome La Sapienza, Department of Chemistry, Rome,
Italy; 4Department of Neurosurgery and Neurotrauma, Ospedale S.
Spirito, Rome, Italy
To increase the knowledge of the static magnetic fields (SMF) and
nerve cells interaction, we investigated the effect of SMF (8-80 mT) on
several primary human glioblastoma cells. The following end-points as
markers of cellular response were tested: apoptosis, oxidative stress,
glutathione depletion, calcium fluxes, plasma membrane integrity and
DNA damage (comet assay). To detect the ability of SMF to modulate
the selected end-points when stress was induced by other chemical/
physical agents, simultaneous exposures were also investigated. The
agents used simultaneously with SMF were: camptothecin, etoposide,
doxorubicin, hyperthermia and x rays. Oxidative stress was monitored
by two different approaches: (i) flow cytometry for cellular ROS
detection; (ii) SOD biosensor for extracellular superoxide anion analysis.
Results: SMF exposure per se did not induce apoptosis, oxidative stress
(very little), and glutathione depletion; but increased the capacitative
calcium fluxes. The glutathione depletion, and ROS production, when
induced by other agents, was not influenced by SMF. It is important to
note that, SMF was able to modulate apoptosis, when triggered by
chemical/physical agents, yielding a lower number of apoptotic figures
in the exposed cells. The extent of induced apoptosis and of SMFmediated rescue were different in the different primary cultures tested,
highlighting an individual sensitivity. We demonstrated by clonogenic
essay that the cells rescued by SMF from chemical/physical triggered
apoptosis, were able to form a progeny. Furthermore, a 24 hour exposure
to 80 mT SMF, interfered with the cell damage induced by other
genotoxic agents, as demonstrated by the comet assay.
109
76
SIMULTANEOUS ANALYSIS OF MULTIPLE CASPASE
ACTIVITIES IN MOUSE T LYMPHOCYTES BY FLOW
CYTOMETRY
William G. Telford1, Beverly Z. Packard2, Akira Komoriya2
1
National Institutes of Health (NIH), Experimental Transplantation
and Immunology Branch, National Cancer Institute (NCI),
Bethesda, Maryland; 2Oncoimmunin, Inc., Gaithersburg, Maryland
78
PRACTICAL STRATEGIES FOR SUCCESSFUL RARE
EVENT DETECTION AND ISOLATION OF HUMAN
HEMATOPOIETIC STEM CELL POPULATIONS WITH
FLOW CYTOMETRIC INSTRUMENTATION
Lora W Barsky1, Ewa Zielinska1, Mary A Price2, Kimberly J
Payne1, Yanjia Zhang1, Qian-Lin Hao1, Yuhua Zhu1, Yasmin
K Parrish1, Kenneth I Weinberg1, Gay M Crooks1
1
Caspases play a critical role as both signaling molecules and effectors in
apoptosis. Cell-permeable fluorogenic caspase substrates have played
an important role in identifying the activation of these proteases in
apoptotic cells; however, fluorochrome and instrument limitations have
generally limited their use to the detection of a single caspase. In this
study, we have employed a series of PhiPhiLux fluorogenic caspase
substrates (developed by Oncoimmunin, Inc.) conjugated to fluorescein, rhodamine- and Cy5-like fluorochromes for simultaneous analysis of
multiple caspase activities. Mouse T lymphoma cell lines were
simultaneously labeled with fluorescein-, rhodamine- and Cy5-like
substrates specific for caspase 1, 3 and 8, and were analyzed on a flow
cytometer equipped with a DPSS 561 nm laser capable of simultaneous
rhodamine and Cy5 excitation and detection. This laser gave excellent
excitation of rhodamine and adequate excitation of Cy5 while not causing
laser light contamination in the fluorescein detector range of conventional
flow cytometers, allowing simultaneous analysis of all three
fluorochrome-linked substrates. Activation of caspase 1, 3 and 8 could
therefore be simultaneously detected, allowing the activation order of
multiple protease activities in early apoptotic cells to be assessed. In
murine T lymphoma cells, caspase 8 activation was found to precede
caspase 3 (as expected from previous biochemical data) and caspase 1
activated simultaneously with caspase 8. This technique allowed
simultaneous temporal mapping of multiple caspase activities in live
cells, information hitherto available only from in vitro biochemical
studies.
Rare Event Detection and Stem Cell
Technologies
77
DETECTION OF MIGRATING FIBROBLASTS IN THE
PERIPHERAL BLOOD BY SLIDE BASED CYTOMETRY
Ulrich Sack1, Christian Eimermacher1, Anja Mittag2, Attila
TáRnok2
1
University of Leipzig, Institute for Clinical Immunology and
Transfusdion Medicine, Leipzig, Saxony, Germany; 2Cardiac
Center, University of Leipzig, Research Laboratory, Pediatric
Cardiology, Leipzig, Saxony, Germany
Rheumatoid arthritis is an inflammatory, chronically proceeding event,
in which aberrant fibroblasts play a crucial role in the pathogenesis and
development of the disease. Our group has recently shown that fibroblasts
isolated from synovial tissue are potential inductors of rheumatoid
arthritis in SCID-Mice. Furthermore, fibroblasts transfected with
interleukin (IL-11, IL-15) have been recovered in joints other than the
one they were injected into, but notably not in other organs. This and
the fact, that rheumatoid arthritis adversely affects several joints
consecutively, leads to the hypothesis that a migration of fibroblasts in
the peripheral blood occurs. We therefore developed a method to isolate
fibroblasts from peripheral blood through means of magnetic beads
(MACS) labelled with a fibroblast-specific antibody (Mab FibAS02),
using an immunomagnetic cell seperation unit (autoMACS). Because of
the rarity of fibroblasts in peripheral blood samples, the cytometric
detection of these cells should be confirmed by cytological methods, in
particular, through use of a Laser Scanning Cytometer (LSC). In contrast
to a conventional Flow Cytometer the technology of the LSC allows a
multi-parametric measurement of adherent cells, including a
relocalization and optical evaluation, and therefore a morphological
assessment of the cells of interest. Thus, it is possible to detect even very
rare cells and, if necessary, to separate them from related populations.
110
Children’s Hospital of Los Angeles, Research Immunology, BMT
and GISCT Program, University Southern California, Los Angeles,
California; 2Barrow Neurological Institute, Neuroimmunology
Research Laboratory, Phoenix, Arizona
Flow Cytometry (FCM) has been used to detect, characterize, and isolate
rare cell populations for twenty years. The availability of sophisticated
multi-parameter FCM has been pivotal for Stem Cell Biology, allowing
the identification and isolation of rare cell types for further analysis.
Technological improvements to flow cytometer instrumentation, such
as high speed and digital electronics, have further facilitated rapid
progress in the field. Nevertheless, isolation of rare hematopoietic stem
cells (HSC) and committed progenitors remains a daunting task for
many laboratories. The cell surface marker CD34 is present on 1% of
human bone marrow and cord blood mononuclear cells and its use in
FCM and magnetic bead separation has proved very useful as an initial
enrichment step for primitive hematopoietic cells. However, the CD34+
population is functionally heterogeneous as it contains HSC and different
types of lineage committed progenitors such as “Common Lymphoid
Progenitors” (CLP). The numerous mature progenitors within the CD34+
population express the CD38 antigen. However, HSC are found in the
1-5% of the CD34+ cells that lack the CD38 antigen (i.e. CD34+CD38-).
This CD34 +CD38- population can be further defined based on CD7
expression. HSC (defined as CD34+CD38-lin-CD7-) are the majority of
the CD34 + CD38- population (and therefore 1 to 5 cells in 10,000
mononuclear cells) whereas, CLP (defined as CD34+CD38-lin-CD7 +)
are 5-10% of CD34 +CD38- cells in cord blood (and therefore <1 in
100,000 mononuclear cells). Meaningful results from functional assays
on HSC and CLP subsets require high cell purity (>99%), which is a
particular challenge considering the initial frequencies of these cells
range from 0.01% to as low as 0.0005% respectively. To achieve the
high purity needed for sensitive functional and PCR assays, our
laboratory protocol requires a series of enrichment steps. Our laboratory
pre-enriches for CD34+ progenitors by magnetic bead separation prior
to FCM sorting. For FCM sorting, we stain to utilize one or more “dump
channels” to remove committed, lineage positive events from the
negative non-committed progenitors, which are further characterized
by specific HSC or CLP markers with remaining fluorescent parameters.
Ultimately, sequential FCM sorting steps are performed to ensure pure
populations. Here we present a discussion of the practical techniques
used for successful detection and isolation of rare and ultra rare
populations from the CD34 + compartment with state of the art
instrumentation and software.
79
INHERENT LIMITATIONS IN RARE CELL DETECTION
Arjan Tibbe1, Craig Miller2, Leon Terstappen2
1
Immunicon Europe Inc., Enschede, Netherlands; 2Immunicon
Corporation, Huntingdon Valley, Pennsylvania
Background. Circulating tumor cells (CTCs) in patients with carcinomas
are extremely rare. In metastatic breast cancer, the presence of >5 CTCs
in 7.5mL of blood has been associated with short survival. As this
threshold has clinical implications, it is important to recognize the
limitations associated with the detection and enumeration of CTCs.
Methods. Poisson statistics was used to determine the probability of
collecting CTCs from a patient in a certain blood volume as a function
of the number of cells in its body. The statistical parameters for sample
processing and final image analysis were empirically determined. All
statistical parameters associated which each step of the process, from
blood collection to final image analysis and CTC enumeration, were
implemented into a model. Using the model a statistical analysis was
performed on data generated from a multi-center clinical trial that utilized
the CellSearchTM System to isolate and enumerate CTCs in 7.5mL blood
samples. Results. The theoretical limit of detection is determined by
the probability of collection a CTC in a 7.5 ml blood sample acquired
from the patient. The blood volume that needs to be acquired to collect
at least one CTC increases very rapidly to volumes that are too large to
acquire without patient suffering. Using the CellSearchTM System we
were able to collect and identify at least one CTC in a single 7.5 ml
blood sample in 35% of the samples, if the total number of CTCs in the
patient´s circulation system was 1000 CTCs and assuming a total blood
volume of 5 liters. Using the model the reader was identified as the
most critical step in the detection of CTCs. The reader will on average
detect 1 CTC in 11% and 2 CTCs in 1% of the cases if no CTCs are
present in the blood sample. In case the reader is error free the threshold
level can be lowered to 1 CTC. This would mean that the presence of
any CTC in the blood would be an identifier for a bad prognosis.
Conclusions. Ultra low detection limits are only possible with a highly
standardized and automated protocol as is used in the CellSearchTM
System. With this system it is possible to detect a single CTC in 7.5 ml of
blood. The amount of blood that can be acquired from the patient is the
limiting factor in reaching lower limits of detection than currently
achieved by the CellSearch TM System.
was 27 (range: 9-53). For panel 2, median CEC/ul was 31(range: 875).. Cells gated as CEC using panel 2 showed an endothelial phenotype
by immunohistochemistry: nucleated cells, lymphocyte-sized and
staining vWF+, CD31+, Fli-1+ and CD34-. CD146 expression was negative
on mature CEC for all tested CD146 mAbs, but positive on HUVECs. In
contrast to PB, analysis of cord blood samples showed a small population
of CD146 + cells, ranging from 0.1-1/ul. Further analysis of this subset
revealed a CD34 +++, VEGFR-2 + phenotype, suggesting a “progenitorlike” function. Conclusions: Our suggested panel and gating strategy
allows enumeration of CEC in PB. Including cells with a higher SSC can
explain an increase in absolute count. Adding a viability stain as in
panel 2 results in detecting viable endothelial cells and allows arbitrary
setting of FSC margins, where as panel 1 does not. Circulating mature
endothelial cells appear in larger amounts than previously accepted by
literature. By detecting rare progenitor-like cells in cord blood, we
have shown that rare event detection by flow cytometry is possible.
81
SP CELLS, ANTHRACYCLINE UPTAKE, AND
FLUORESCENCE POLARIZATION
Timothy W Petersen1, Allan Kachelmeier2, Sherrif Ibrahim3,
Ger Van Den Engh4
1
Cytopeia, Inc., Seattle, Washington; 2Oregon Health Sciences
University, Portland, Oregon; 3University of Washington Medical
Center, Seattle, Washington; 4University of Washington,
Oceanography, Seattle, Washington
The graph displays the probability of collecting >= 1 CTC in one
out of n = 1, 2, 3 or 4 samples, each 7.5 ml as a function of the
number of CTCs in vivo assuming a blood volume of 5 liters. 1
CTC / 7.5 ml corresponds to 667 in vivo CTCs.
80
UNEXPECTED HIGH COUNTS OF MATURE
CIRCULATING ENDOTHELIAL CELLS IN HEALTHY
INDIVIDUALS
Michiel Strijbos1, Jaco Kraan1, Michael Den Bakker2, Bart
Lambrecht3, Stefan Sleijfer1, Jan-Willem Gratama1
1
Erasmus Medical Center, Medical Oncology, Rotterdam, ,
Netherlands; 2Erasmus Medical Center, Pathology, Rotterdam, ,
Netherlands; 3Erasmus Medical Center, Pulmonary Medicine,
Rotterdam, , Netherlands
Background: Circulating endothelial cells (CEC) are shed from damaged
vasculature making them a rational choice to serve as surrogate marker
for drug efficiency, and changes in CEC in peripheral blood (PB) might
predict therapy response or disease progression. The aim of our study
was to develop an optimal antibody panel and gating strategy by which
absolute CEC counts can be robustly and reproducibly acquired using a
three-color flow cytometer. Methods: PB from 40 healthy donors (age
23-70), was stained in a stain-lyse-no wash procedure using two panels
of monoclonal antibodies: panel 1: CD31-FITC, CD45-PerCP and panel
2: CD31-FITC, CD45-PE, 7-AAD. CEC were defined as
FSCintermediate, SSClow, CD45dim and CD31bright. For panel 1, an
FSC-SSC plot was set roughly around leukocytes to exclude debris and
platelets. Panel 2 data was gated after exclusion of dead cells (7-AAD+).
Absolute counts were obtained using fluorescent counting beads added
to each staining. The endothelial origin of CEC was confirmed by cell
sorting followed by immunohistochemistry for von Willebrand factor
(vWF), CD31, CD34 and Fli-1 antibodies. Three different CD146 mAbs
were tested on CEC, HUVECs and a cord blood. Results: With our
suggested method, mature endothelial cells can be enumerated in whole
blood without enrichment. Our absolute count yields much more
endothelial cells when compared with techniques based on enrichment
or even other flow cytometrical approaches. Median CEC/ul for panel 1
When bone marrow cells are stained with Hoechst 33342 dye, a small
group of cells distinguishes itself because of a spectral shift to shorter
wavelengths. These so-called Side Population (SP) cells appear to harbor
most, if not all of the stem cell activity 1. Petersen et al.2 have shown
that the color of Hoechst fluorescence is related to the cellular dye
concentration. Because side population cells have a low uptake rate,
they take longer to make the blue-to-red spectral shift than most cells.
Fluorescence polarization studies 3 show that the color variation of
Hoechst is due to resonant energy transfer between closely spaced
molecules. In cells with a high dye concentration, the molecules sit
closely together and dye-dye interactions cause a red shift of the
fluorescence. In addition to showing a red shift, the emitted fluorescence
is also depolarized as a result of the resonant energy transfer. Most
DNA dyes do not exhibit noticeable color shifts with increasing
concentration. However, polarization changes with energy transfer are
readily observed. If the two phenomena are linked, fluorescence
polarization could be used as an alternative to shifts in fluorescence
color. If that postulation is true, one should be able to use most viable
DNA stains to mark SP cells. We will present results of experiments that
correlate the uptake of the anti-cancer Anthracycline drugs polarization
and show that this parameter can be used to distinguish between different
subpopulations in mouse bone marrow cells. 1.
Ibrahim SF, Diercks
AH, Petersen TW, van den Engh G (2005) All bone marrow cells
transiently exhibit the side population (SP) phenotype. Exp.Hematol.
submitted.
2.
Petersen TW, Ibrahim SF, Diercks AH, van den EG
(2004) Chromatic shifts in the fluorescence emitted by murine
thymocytes stained with Hoechst 33342. Cytometry 60A:173-181.
3.
Uy JL, Asbury CL, Petersen TW, van den Engh G (2004) The polarization
of fluorescence of DNA stains depends on the incorporation density of
the dye molecules. Cytometry 61A:18-25.
82
FLOW CYTOMETRIC ANALYSIS AND PURIFICATION
OF NEURAL AND NEURONAL CELL POPULATIONS
DERIVED FROM HUMAN EMBRYONIC STEM CELLS
Jan Pruszak1, Kai-Christian Sonntag1, Joris Van
Arensbergen1, Takahito Yoshizaki1, Moe Hein Aung1,
Rosario Sanchez-Pernaute1, Ole Isacson1
1
Center for Neuroregeneration Research, Harvard Medical School,
McLean Hospital, Belmont, Massachusetts
Human embryonic stem cells (hESC) represent a promising cell source
to generate functional neurons for future cell therapy of
neurodegenerative diseases. One problem of current in vitro
differentiation protocols is the development of heterogeneous cell
populations. However, a defined cellular composition and high purity
of the cell suspension are required for potential clinical applications.
Here, we adapt a range of flow cytometric methodology for analysis
111
and cell selection of hESC-derived neural and neuronal cells.
Analytically, we used intracellular staining for the glial fibrillary acidic
protein (GFAP), the intermediate filament Nestin, neuron-specific class
III beta-tubulin and for tyrosine hydroxylase to identify astroglial, neural
precursor, neuronal and dopaminergic cells, respectively, by flow
cytometry after fixation. Proliferative populations in cultured hESC
were detected by bromodesoxyuridine (BrdU)-labeling, and we used
viability assays with propidium iodide, caspase-3 and annexin-5, as
well as indicators of oxidative metabolism, e.g. CM-H 2 DCFDA, for
neuronal toxicity and vulnerability studies. For cell selection, we tested
surface antigens for their potential to purify subpopulations at the
immature, precursor and neuronal stage by fluorescent-activated cell
sorting (FACS), or alternatively magnetic cell separation. Purified hESC
were cultivated post-sort and characterized for parameters relevant for
neurogenesis and neuronal specification using immunocytochemistry,
RT-PCR, BrdU-assays and transplantation studies. Undifferentiated hESC
were enriched for the stage-specific embryonic antigen SSEA-4, and
immaturity was confirmed by positivity for TRA-1-81, TRA-1-60 and
SSEA-3. Neuroectodermal precursors were characterized by surface
antigens like SSEA-1, FORSE-1, and NCAM (CD56) and sorted for
further differentiation in vitro. Differentiated cells were analyzed and
FACS-purified for differential expression of p75 (CD271), NCAM and
CD24. The latter led to the isolation of two Nestin-positive
subpopulations, CD24 high and CD24 low , with distinct growth and
differentiation patterns. Transplantation of NCAM-positive
subpopulations into a rat model of Parkinson´s disease showed postsort survival of neural cells in vivo. Our results demonstrate how flow
cytometry can be applied for analysis and cell selection of hESC-derived
neural and neuronal cells.
Image processing and Analysis 3
83
DETECTION AND ASSESSMENT OF CERVICAL
INTRAEPITHELIAL NEOPLASIA (CIN) LESIONS BY
DNA IMAGE CYTOMETRY
Sun Xiaorong1
1
Canada BC Cancer Agency;Wuhan Landing Early Cancer Detecting
Center, Wuhan, Hubei, China
Objectives: To compare detection and prognostic assessment of CIN
lesions between conventional cytology and DNA image cytometry (DNAICM) assisted cytology. Methods: The study enrolled 87 women. Cervical
samples were collected employing cervix brushes which were then
washed in SedFix. Two slides were prepared from each case: one slide
was stained by Papanicolaou stain for conventional cytology, while the
other slide was stained by Feulgen-Thionin method for measurements
of the amount of DNA in the cell nuclei using an automated DNA
imaging cytometer. Results: Of 87 cases 30 were called normal by
conventional cytology. Of the total of 20 ASCUS cases called by
conventional cytology, no CIN2 or greater lesions were found in the 7
cases that did not contain any cells with DNA amount greater than 5c,
while CIN2 lesions were found in 11 out of 13 cases that had some
aneuploid cells with DNA amount greater than 5c. Of 30 LSIL cases
called by conventional cytology, CIN2 lesions were detected in 3 out of
7 cases that did not contain any aneuploid cells with DNA greater than
5c and in 22 out of 23 cases that contained aneuploid cells with DNA
amount greater than >5c. Of the residual 7 cases that conventional
cytology called HSIL, all case contained aneuploid cells containing
DNA greater than 5c. If cytology were to be used to refer all cases of
LSIL and HSIL to colposcopy and biopsy procedure to detect potential
CIN2 or greater lesions then sensitivity, specificity, positive predictive
value and negative predictive value would have been 58%, 84%, 87%
and 54%, respectively. If DNA-ICM were to be used instead, and all
cases having 3 or more cells with a DNA amount greater than 5c were to
be referred to colposcopy and biopsy to detect potential CIN2 or greater
lesions then sensitivity, specificity, positive predictive value and negative
predictive value would have been 73%, 88%, 90% and 65%, respectively.
We also compared Ki67 positivity in these samples and found that
DNA-ICM results are comparable to this biomarker method. Conclusions
This study demonstrates that DNA-ICM can be successfully used to
detect significant (i.e. CIN2 or greater) lesions. It has been shown before
that the presence of aneuploid cells containing DNA amount greater
than 5c strongly predicts a progressing lesion that would lead to an
invasive cancer unless treated. Therefore, the DNA-ICM approach
provides also a prognostic assessment of CIN lesions.
112
84
CELL SIZE, SHAPE AND MEMBRANE ARE TARGETS OF
STATIC MAGNETIC FIELDS AS DEMONSTRATED BY
ELECTRON, OPTIC AND ATOMIC FORCE MICROSCOPY
IN HUMAN GLIOBLASTOMA CELLS
Laura Teodori1, Maria Cristina Albertini2, Francesco
Uguccioni2, Elisabetta Falcieri3, Marco Rocchi4, Antonio
Bergamaschi5, Andrea Magrini5, Raffaele Mucciato6,
Augusto Accorsi2
1
ENEA - Casaccia, BIOTEC-MED, Rome, Italy; 2University of
Urbino, Institute of Biochemistry G. Fornaini, Urbino, Italy;
3
University of Urbino, Institute of Morphological Sciences, Urbino,
Italy; 4University of Urbino, Istitute of Biomedical Engineering,
Urbino, Italy; 5University of Rome ‘Tor Vergata’, Occupational
Health, Rome, Italy; 62M strumenti, Rome, Italy
Background. There are very few theoretical reasons which support that
static magnetic fields (SMF) might cause or contribute to cancer or any
other human health problems and there is very little laboratory or
epidemiological evidence that connects SMF exposure and human health
hazards. However, some experimental results demonstrated that SMF
affect cellular structures and functions. In order to investigate the effect
of exposure to increasing doses of SMF on cell morphology, human
glioblastoma cells were exposed to SMF ranging between 80 and 3,000
G. Cell morphology was studied by electron, optic and atomic force
microscopy. Results. Optical microscope images showed altered
orientation and alignment of exposed cells as demonstrated by circular
statistic analysis. TEM images of the 3,000 G treated cells revealed the
presence of micronuclei and dense vacuolized cytoplasm. The
modifications at 80 and 300 G were not remarkable. Scanning electron
microscopy demonstrated a dose dependent cell shape and size
modification as demonstrated by the roundness index. Loss of the long
villi, and appearance of membrane roughness and blebs were also
evident. The cellular actin distribution detected by FITC-phalloidin
staining showed that SMF exposure produced actin filament contraction.
The atomic force microscopy of the exposed cells demonstrated
membrane reorganization and several significant dose dependent
modifications such as disappearance of the ordered surface ripples and
furrows typical of the unexposed cells and occurrence of surface
membrane corrugation. The roughness index was also affected (p =
0.009) Conclusions. Our experimental procedures demonstrated that
exposure to SMF affects size, shape orientation and membrane surface
of glioblastoma cells.
85
IMAGING THE CELLULAR RESPONSE TO FLUID
SHEAR BY APPLYING ULTRA-LOCALIZED FLOW
FIELDS GENERATED BY ROTATING LASER-TRAPPED
MICROSPHERES
Elliot Botvinick1, Gregor Knoener2, Michael W. Berns3,
Halina Rubinsztein-Dunlop2
1
Beckman Laser Institute, Irvine, California; 2University of
Queensland, Physics, Brisbane, Queensland, Australia; 3University
of California, Irvine, Surgery, College of Medicine, Irvine,
California
An advanced microscope system has been developed to measure spatial
and temporal responses to fluid shear applied to local regions of the cell
surface. The microscope is built on the RoboLase platform and
incorporates fluorescent and wide filed imaging as well as Florescent
Resonance Energy Transfer (FRET) measurements. The microscope
also incorporates the laser-mediated tools of Fluorescence Recovery
after Photobleaching (FRAP), laser ablation and laser tweezers. As a
new ‘spin´ on applying fluid shear to the cell surface, spherical vaterite
crystals are rotated near the cell surface to apply flow rate gradients
generally confined to the length scale of the particle diameter. The
crystals are held in a 3-D laser trap of circularly polarized light that
applies optical torque to the crystal thus rotating it with respect to the
surrounding liquid media. The optical torque can be quantified optically
by measuring the polarization change of light passing through the crystal
to provide a direct measure of the drag torque. Crystals a few microns
in diameter can be rotated at hundreds of Hertz thus applying
physiological shear stresses to confined regions of the cell surface. This
mechanism of interaction allows the study of heterospatial force
transduction and force-mediated cell properties. Furthermore this
method provides an uncoupled control for complementary ligand
coupled bead-pulling experiments, as the fluid-coupled stresses do not
directly tie into the cytoskeleton, surface molecules or the cortical actin.
By optically monitoring the laser power, rotation rate, and polarization
change, the environment surrounding the crystal can be monitored for
changes which may provide new insight as to the extent and role of
surface molecules as they pertain to flow-induced function.
86
APPLICATION OF QUANTITATIVE MORPHOLOGICAL
CYTOMETRY FOR EVALUATION OF SHEAR STRESS
Dominik Lenz1, Bulent Bayraktar2, Silas Leavesley1, J. Paul
Robinson3, Bartlomiej Rajwa1
1
Indiana; 2Purdue University, Electrical and Computer Engineering,
Schools of Engineering, West Lafayette, Indiana; 3Purdue
University, Basic Medical Sciences & Biomedical Engineering, West
Lafayette, Indiana
Background: Shear stress is well known to significantly affect the state
of cellular differentiation, protein expression, and shape. The influence
of shear stress on differentiation has been thoroughly evaluated.
However, shear-induced changes in morphology have yet to be
quantified objectively using shape descriptors. The descriptors are
numbers that describe inherent features of a given shape, and can be
used for classifying, matching and recognizing objects. The goal of this
study was to find shape parameters (descriptors) that could be correlated
with the amount of shear stress, and which could be used to distinguish
between cells exposed to low and high shear stress conditions. Methods:
Bovine aortic endothelial cells (BAEC´s) were exposed to varying levels
of shear stress (2, 15, and 30 dynes) for a period of 24 hours. The
specimens were imaged using a high-content screening system.
Randomly chosen 128 cells from each group were segmented and used
to calculate a set of shape descriptors, such as form factor, roundness,
aspect ratio, convexity, solidity, compactness, and extent. Additionally,
three parameters based on geometrical moments (elongation, dispension,
and extension), as well as Fourier boundary descriptors were computed.
Results: Representative shape descriptors and geometrical moments
showed high degree of correlation to the amount of shear stress. In
contrast, Fourier descriptors did not show monotonous dependence on
shear stress. Interestingly, multiparameter discriminant analysis
demonstrated that traditional shape descriptors were more useful for
discriminating between shapes of cells belonging to three different groups
than more complex moment-based parameters. Conclusion: The results
of this study showed that relatively simple and easy-to-compute shape
descriptors can be employed for the purpose of quantifying the influence
of shear stress on cellular morphology. It is likely that other types of
environmental stress may also result in quantifiable changes in cellular
morphology. The tools presented in this manuscript can be used to
extract additional shape-related parameters during the process of slidebased cytometry analysis or high-content screening. Key words:
endothelial, aortic, morphology, shape analysis, shear, stress, slide-based
cytometry, high content screening
Subcellular
Location
Image
Database
(PSLID,
http://
murphylab.web.cmu.edu/services/PSLID) is an open source database
software package for comprehensive storage of high resolution FMI
and automated analysis of the images [K. Huang et al, Proc. 2002 IEEE
Intl Symp Biomed Imaging, pp. 325-328]. The current schema of the
database makes it possible to manage the detailed experimental
information on image data collection, the high resolution FMI of 2
dimensions through 5 dimensions and the subcellular location features
(SLF) of these image objects for interpreting the subcellular location
patterns of the target proteins. A web-based interface has also been
implemented to provide comprehensive image search and analysis
capabilities. The search of the image can be either based on context (the
annotation of the imaging experiments) or content (the numerical SLFs
extracted from the images). The image analysis module of PSLID
implements many tools which have been developed in our group over
the past ten years [R.F. Murphy, Cytometry 67A:1-3]. It includes
statistical inferences based on image content such as typical image
selection (TypIC) and set comparison (SImEC), and machine learning
algorithms for both classification and clustering. These tools have been
shown to provide better sensitivity and accuracy than human visual
inspection. PSLID is built on the Postgres database system and the
Tomcat Java Server Page server on the Linux platform, both of which
are open source. It is part of a collaborative Information Technology
Research project joint with the University of California Santa Barbara
and funded by the National Science Foundation. Current work is aimed
at providing an interface between PSLID and the OME database system
developed by the Open Microscopy Environment project (http://
www.openmicroscopy.org).
88
CELLPROFILER: FREE, HIGH-THROUGHPUT
SOFTWARE FOR AUTOMATICALLY MEASURING
CELLS IN IMAGES
Anne E. Carpenter1
1
Whitehead Institute for Biomedical Research, Sabatini Lab,
Cambridge, Massachusetts
Advances in imaging hardware now allow the rapid collection of
thousands of high resolution images of cells. Automatically measuring
features of cells quantitatively from these images has been difficult due
to the limitations and often proprietary nature of available image analysis
software. We have therefore developed CellProfiler cell image analysis
software to allow biologists without training in computer vision or
programming to quantitatively measure cells in thousands of images
automatically, without tedious user interacion. This freely available,
open-source software project is modular and compatible with most
image formats and movie formats, allowing adaptation to a variety of
cell types and assays. We have tested the software using cells from
human, mouse, yeast, and fruit fly to measure phenotypes including
cell count, cell size, cell cycle distribution, and the levels and localization
of proteins and phospho-proteins, including application to time-lapse
and high-throughput experiments.
87
PROTEIN SUBCELLULAR LOCATION IMAGE
DATABASE FOR COMPREHENSIVE IMAGE RETRIEVAL
AND AUTOMATED INTERPRETATION
Juchang Hua1, Ting Zhao2, Shann-Ching Chen3, Yanhua
Hu1, Amol Shanbhag3, Justin Newberg3, Swapnil
Upganlawar1, Robert F. Murphy2
1
Carnegie Mellon University, Department of Biological Sciences,
Mellon College of Science, Pittsburgh, Pennsylvania; 2Carnegie
Mellon University, Department of Biomedical Engineering, Carnegie
Institute of Technology, Pittsburgh, Pennsylvania; 3Carnegie Mellon
University, Department of Medical Engineering, Carnegie Institute
of Technology, Pittsburgh, Pennsylvania
Computational analysis of high resolution fluorescence microscope
images (FMI) permits the automated, accurate, objective and sensitive
determination of protein subcellular locations. The growing volume of
images collected for this purpose require a database system which can
efficiently maintain the image datasets, facilitate retrieval, and, most
importantly, enable automated and sophisticated analysis. The Protein
113
BioPharma Applications
89
DEVELOPMENT OF MULTILAYERED NANOPARTICLE
SYSTEMS FOR NANOMEDICINE
generation times after drug exposure, which facilitates work with slower
growing organisms such as Helicobacter pylori and Mycobacterium
tuberculosis . Cytometry also permits detection of small numbers of
resistant organisms, and has pointed the way toward development of
new combination therapies based on selectively increasing permeability
of bacteria to otherwise nontoxic agents. Portions of this work were
supported by NIH Grant AI063833.
James F. Leary1, Tarl W Prow2, Donald EUGENE Bergstrom3
1
Purdue University, Basic Medical Sciences, West Lafayette,
Indiana; 2Johns Hopkins University, Baltimore, Maryland; 3Purdue
University, Medicinal Chem & Molecular Pharmacology, Pharmacy
& Pharmacal Sciences, West Lafayette, Indiana
Nanomedicine provides for a revolutionary new approach to regenerative
medicine by a “bottom up” approach whereby tissues and organs are
treated in parallel processing fashion at the single cell level using billions
of smart, multilayered nanoparticles. Such nanoparticle drug delivery
systems have immense promise for more accurate delivery of drugs
with a lower rate of bystander cell mis-targeting. Such enclosed
nanosystems also protect drugs from being broken down in the
bloodstream, a common problem in drug delivery. By employing a
multi-step process, the nanosystem can contain built-in error-checking
to minimize bystander effects. Targeting can also be intracellular in
order to bring a drug into close proximity of its molecular target inside
single living cells. Since these nanosystems contain both diagnostic and
therapeutic features, the conventional distinction between diagnostics
and therapeutics is blurred. A new term “theragnostics” describes this
new concept of simultaneous diagnostics and initial therapeutics.
Therapeutic drugs or genes can be delivered inside single cells whereby
the dose is controlled by biomolecular sensors on a cell-by-cell level.
Multilayered nanoparticle systems are being constructed on a variety of
core particle types (nanogold, quantum dots, paramagnetic). The
nanosystems are built in a layer-by-layer assembly and are designed to
disassemble layer-by-layer in reverse order in-vivo effectively creating
a programmable nanoparticle system (Leary and Prow, patent pending).
Nanosystems from 60 – 160 nm diameter are probably optimal for invivo therapeutics.
Nanoparticle targeting to cells can be assessed
using a combination of MACS magnetic sorting, fluorescence membrane
tracking probes, fluorescence microscopy, flow cytometry and LEAP
scanning cytometry/laser optoinjection. By using PCR amplifiable
sequences, biodistribution of these nanoparticle systems can be studied
in animals even when the nanoparticles in tissues cannot easily be located
for conventional imaging techniques.
The applications of this
platform nanotechnology are numerous. Specific examples for cancer
and antiviral treatments will be discussed.
90
CYTOMETRY AS AN AID IN DEVELOPMENT OF
ANTIMICROBIAL AGENTS
Howard Shapiro1, Nancy Perlmutter2
1
2
Howard M. Shapiro, M.D., P.C., West Newton, Massachusetts;
Howard M. Shapiro, M.D., P.C., Allston, Massachusetts
The effects of antimicrobial agents on microorganisms are almost always
assessed at the population or colony level. Modern instruments for
determination of drug susceptibility typically measure increases in
bacterial mass, as indicated by increasing turbidity of liquid media, or
increases in products of bacterial metabolism, as indicated by reduction
of tetrazolium dyes, changes in the pH or impedance of the medium,
etc. Even when response to antibiotics is assessed by colony counts of
treated and untreated cultures, essentially no information is obtained
about changes in the physiology of individual organisms. Although
modern multiparameter flow cytometry allows cell-by-cell analysis of
the kinetics of changes in several physiological characteristics associated
with the transition from a viable to a non-viable state, most studies of
the interactions of antimicrobials and bacteria or fungi have measured
only a single characteristic, typically membrane permeability or
membrane potential. More recent studies, in which refined ratiometric
methods were used for simultaneous measurement of both parameters,
show that cell dynamics following exposure to antimicrobials may be
more complex than initially expected, and that different species and
strains may respond differently to different antibiotics. Although this
complexity may preclude the development of simple, rapid cytometric
clinical assays for drug susceptibility, it allows multiparameter cytometry
to provide some otherwise unobtainable insights into mechanisms of
antimicrobial action at the single cell level, usually within a few
114
91
DEVELOPMENT OF A PHOSPHATIDYLINOSITOL 3KINASE DRUG ACTIVITY BIOMARKER ASSAY IN
PLATELETS
Rita Bowers1, Philip Marder1, Lisa Green1, Andrew Faber1,
Phillip Schwier1, Candice Horn1, James Thomas1
1
Indianapolis, Indiana
The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a
pivotal role in controlling proliferation, motility, and survival of cells.
Recent reports have indicated that >30% of human colorectal, breast,
and hepatocellular cancers display somatic mutations in the alpha isoform
of PI3K. This (and other evidence) has provided a rationale for clinical
development of new drugs that might block such aberrant PI3K activity.
In order to bring targeted agents to market more quickly, development
of selective drug activity biomarker assays has become essential for
preclinical and early phase human studies. An ideal drug activity
biomarker should be robust, reproducible, easily attainable, and closely
linked to the molecular target of interest. Platelets contain a rich source
of PI3K (including the alpha isoform) that can be activated through
stimulation of protease-activated receptors (PARs) using thrombin or its
derived peptide ligands. Once stimulated, this upstream PI3K activity
activates platelets into a cascade of signaling events including
conformational changes in the fibrinogen receptor. In this study, we
used flow cytometry to measure downstream PAR-induced platelet
activation as a surrogate biomarker for modulation of PI3K activity in
tumors. Results described in this presentation reveal a robust (10 – 30
fold fluorescence increase) signal that was blocked by the selective
PI3K inhibitors, Wortmannin (WORT) and LY294002 (LY). The assay
is functional in both human and mouse systems. For human sample
analysis, platelets are isolated using size exclusion chromatography and
assayed in citrated plasma by activation through PAR-1 followed by
reaction with PAC-1-FITC monoclonal antibody. For the mouse, platelets
are activated in whole blood through PAR-4 and reacted with alexafluor-488-fibrinogen. Both human and murine systems were blocked
(in vitro) by WORT and LY in a concentration dependent manner.
Furthermore, mice were dosed intraperitoneally with WORT (4 mg/kg)
or vehicle and citrated blood was collected after 1 hr. and tested (ex
vivo) for PAR-4-induced activation. Significant inhibition of PAR-4induced platelet activation was observed in WORT treated normal and
tumor-bearing mice. Results from these studies indicate that flow
cytometric measurement of PAR-induced platelet activation could be a
suitable drug activity biomarker assay for evaluation of putative PI3K
inhibitors in the clinic.
92
QUANTITATIVE ANALYSIS OF THERAPEUTIC
MONOCLONAL ANTIBODY LOCALIZATION TO
ENDOSOMES AND LYSOSOMES USING THE
Brian Hall1, Philip Morrissey1, Che-Leung Law2, Kristine
Gordon2, David Lynch1, Keith Frost1, Thaddeus George1,
David Basiji1, Cathleen Zimmerman1, William Ortyn1,
Richard Bauer1, David J Perry1, Richard Esposito1
1
Amnis Corp., Seattle, Washington; 2Seattle Genetics, Bothell,
Washington
Monoclonal antibodies (mAb) have recently become clinically effective
drugs for the treatment of a variety of human diseases. A number of
these bind to cell surface determinants and thus are subject to cellular
mechanisms of membrane clearing which include internalization into
the endosomal/lysosomal degradation pathway. However, this pathway
is complex and sorting to different compartments and recycling are
known to exist. The efficacy of therapeutic mAb could be influenced
by the rate of membrane clearing and progression to the degradative
lysosmal compartment. Monoclonal antibodies used as single agents
may be less effective if cleared rapidly from the cell surface since there
may be less chance to fix complement or act as an opsonin. Alternatively,
mAb conjugated to chemotherapeutic agents may need to access the
lysosomal compartment in order to become active. One such mAb used
clinically, binds to CD20 which is expressed on chronic lymphocytic
leukemia cells. Interestingly, not all patients have a therapeutic response
when treated with anti-CD20 (Rituximab). Conceivably, the difference
may be due to the clearance and catabolism of the mAb amongst patient
populations. Thus it would be of interest to determine the cellular fate
of the mAb in established tumor cell lines and eventually apply this
analysis to patient samples. A proof of concept study to track antibody
drug conjugate trafficking has been performed utilizing the ImageStream
imaging flow cytometer which acquires 6 channels of imagery
simultaneously from cells in flow including bright field, dark field and
4 channels of fluorescent imagery at acquisition speeds of 300 cells per
second. The Ramos Burkitt lymphoma cell line was incubated with
fluorochrome labeled anti-CD20 for various times and then fixed and
stained with fluorochrome labeled markers for endosomes and
lysosomes. Cellular imagery was acquired and was analyzed using an
algorithm that compares the similarity of the fluorescent imagery on a
pixel by pixel basis between channels in a quantitative manner using the
non-mean normalized Pearson´s correlation coefficient. The data
demonstrated an initial capping of the anti-CD20, followed by
association with the endosome marker and later with the lysosome
marker. This was observed in sample sizes of 10,000 cells with a
quantitative score which allows for statistical analysis. Thus this approach
should be useful clinically in analyzing potential differences in
intracellular fates of mAb between responder and non-responder
populations.
93
DEVELOPMENT OF HIGHLY-SECRETING
BIOPHARMACEUTICAL CELL LINES VIA IN SITU
MEASUREMENT OF CELL-SPECIFIC ANTIBODY
SECRETION, LASER-MEDIATED CELL PURIFICATION,
AND AUTOMATED CLONE TRACKING AND ANALYSIS
Elie Hanania1, Janine Stevens1, Gary Bright1, Manfred
Koller1
1
Cyntellect, San Diego, California
Development of highly-secreting recombinant cell lines is critical for
efficient biopharmaceutical manufacturing of many important
therapeutic proteins. Currently used approaches are low-throughput,
involve significant labor for clone tracking and analysis, and do not
account for the fact that secreted protein does not remain associated
with the best-producing cells. A fundamentally new approach was
developed combining in situ capture and measurement of individual
cell protein secretion followed by laser-mediated elimination of all nonand poorly-secreting cells, leaving only the highest-secreting cell in a
well. Recombinant cells producing humanized antibody were cultured
serum-free on a capture matrix in 96- and 384-well plates, followed by
staining with fluorescently-labeled anti-human antibody fragment. A
novel, automated, high-throughput instrument (called LEAP™) was
used to image each well (containing up to 10,000 cells), quantify the
cell-associated and secreted antibody surrounding each cell, and eliminate
all undesired cells from a well via targeted laser irradiation (at the rate of
>1,000 cells per second). Temporarily sparing an island of helper cells
within the well improved cloning efficiency (particularly when using
serum-free medium), and the helper cells were easily eliminated with
the laser after several days. Cell number was automatically tracked
using brightfield cell counting within well plates at various time points
after cloning, allowing efforts to be focused on truly clonal wells
containing clones with the best growth properties. The automated in
situ capture assay was also employed after cloning to evaluate
productivity from a few hundred cells, allowing poor clones to be
discarded before significant cell culture expansion effort was expended
leading to standard assays requiring millions of cells (e.g., ELISA and
HPLC). The in situ nature of this process allowed several serial subcloning steps to be performed within days of one another, resulting in
rapid generation of clonal populations with significantly increased and
more stable, homogeneous antibody secretion.
94
DETERMINATION OF IN VIVO TOXICITIES OF GAMMA
-SECRETASE INHIBITORS IN SPLEEN MARGINAL
ZONE B CELLS BY FLOW CYTOMETRY
Sharon Ann Sokolowski1, Barbara-Anne Martin2, Anne M.
Ryan3, Gary B. Freeman4, Carol D. Hicks2, Lit-Fui Lau2,
Nikolay Pozdnyakov2
1
Pfizer PGRD, Groton, Connecticut; 2Pfizer PGRD, CNS
Discovery, Groton, Connecticut; 3Pfizer PGRD, Pathology, Groton,
Connecticut; 4Pfizer PGRD, Toxicology, Groton, Connecticut
Beta-Amyloid is a major component of the senile plaque in Alzheimer’s
disease (AD). It is believed to be the main culprit in AD pathogenesis
according to the amyloid cascade hypothesis. Its production requires
the concerted proteolysis of amyloid precursor protein (APP) by both
BACE and -secretase. Inhibition of -secretase, therefore, represents a
plausible approach to treat AD. However, in addition to APP, gammasecretase cleaves a number of other substrates; one of these is Notch.
Notch signaling is important in diverse cellular and developmental
processes, including differentiation, proliferation, survival and apoptosis.
Consistent with the inhibition of these Notch-dependent processes, some
gamma-secretase inhibitors have been shown to cause intestinal Goblet
cell metaplasia, and depletion of lymphocytes. Selectivity of -secretase
inhibitors over Notch processing becomes a key safety goal. Here, we
have successfully monitored depletion of marginal zone B cells using
fluorescence-activated cell sorting (FACS) technique.
95
NON-INVASIVE AND LABEL-FREE FLOW CYTOMETRY
Marco Di Berardino1, Grit Schade1, Adrian Huwiler1, Thomas
Hessler1
1
Leister Process Technologies, Microsystems, Kaegiswil,
Switzerland
Flow cytometry has become a routine technique for cellular analyses.
However, this technology is still very complex and usually requires
costly colour reagents as well as specifically trained people for its
operation. Many efforts have been put in the past few years into the
development of easier and less expensive systems. Innovative
technologies with alternative approaches emerged aiming at reducing
cost of consumables and at simplifying whole analyses processes. In
this context, various microfluidic devices have been developed and
many chip-based flow cytometers investigated for biological applications.
Beside the integration of traditional optical detection methods relying
on fluorescent reagents mainly electrical measurement techniques were
pursued. However, these developments had primarily academic
relevance, were rather used for size discrimination purposes and ended
by showing the proof-of-concept only. We went a step ahead and present
here an improved device for cell analysis and characterization
applications. The chip-based flow cytometer relies on the measurement
of the electrical properties of cells flowing through a microfluidic channel.
Impedance spectroscopy provides information on cell size, membrane
capacity and cytoplasm conductivity. Issues of non-physiological
conditions or hydrodynamic stress are overcome by applying
dielectrophoresis within the microfluidic chip. In-flow single cell
measurements in our microchip are possible without extensive sample
preparation or addition of specific markers or dyes. Discrimination of
various cell line types, such as undifferentiated mouse fibroblasts (3T3)
and adipocytes, could be easily achieved. Impedance measurements of
monocytes and differentiated monocytes to dendritic cells and
macrophages indicate that a distinction of these cells is also possible.
The ability to separate blood cell types as lymphoblasts, granulocytes
and monocytes emphasizes the suitability of the system for many other
haematological applications. For some cell models also viability and
apoptosis measurements were carried out successfully. Finally, analyses
of several species from yeast, bacteria and fungi not only demonstrate
the ability to enumerate these cells, but also showed that even sporulation
and other microbiological live cycle phases can be visualized. In
summary, this microfluidic approach combines the advantages of
performing assays in small sample volumes with minimal sample
preparation efforts and without the need of any cell labelling. The
device, which is also designed for cell sorting applications, represents a
powerful pre-diagnostic cell analysis tool and is a valuable complement
to the known and rather expensive fluorescence-based flow cytometers.
115
96
MULTISPECTRAL CYTOMETY: A POWERFUL NEW
TECHNOLOGY IN CELL ANALYSIS
J. Paul Robinson1, Bartlomiej Rajwa2, Kathy Rajheb3, James
T. Jones4, GéRald GréGori5, Valery P. Patsekin3
1
Engineering, West Lafayette, Indiana; 2Purdue University, Basic
Medical Sciences, Veterinary Medicine, West Lafayette, Indiana;
3
Purdue University, Bindley Bioscience Center, W. Lafayette,
Indiana; 4Purdue University, Institute-Interdisciplinary Engr Studies
- Biomed. Engr. Ctr., Engineering, West LaFayette, Indiana;
5
Université de la Méditerranée, Marseille, Marsailes, France
analysis of more discrete emission bands within a given wavelength
range, reduce electronic crosstalk, and allow more precise resolution of
the relative contribution of individual fluorophores in multiply-tagged
samples thereby enabling a range of new applications involving the
spectral analysis of single cells and particles.This effort supported by
NIH RR020064-01, and NIH RR001315-23 funding.
98
RAMAN FLOW CYTOMETRY
John P. Nolan1, Dakota Watson1, Daniel Gaskill1, Mirianas
Chachisvilis1, Steven Graves2, Lief Brown3, Stephen Doorn3,
Hicham Fenniri4
1
We have developed a flow cytometer with the capability of collecting
32 channels of fluorescence spectra enabling a quantum leap in flow
cytometry systems. Unlike previous instruments where single
photomultiplier tubes collected wide bands of spectral bands, this
instrument enables us to transform the entire spectrum of a particle of
cell as a spectral parameter. This is a major change in technology and
opens up many new possibilities including the use of multiple dyes with
almost totally overlapping spectra as well as effectively removing the
need for traditionally complex spectral compensation. There are many
advantages of this technology that may not be obvious. The first is the
vast reduction in hardware – this system utilizes a single multiplexed
PMT with a single board for all the data collection electronics. Second,
because we collect the entire spectrum, we can utize advanced algorithms
to classify the spectrum in a variety of classes enabling us to classify
cells or particles semi-automatically. Third, the opportunity now arises
for very advanced databasing and multivariate analysis of data. This
latter point is very important because the great majority of flow cytometry
data, regardless of the number of variables collected is analyzed using
univariate or bivariate techniques. When one has 32-40 variables,
traditional analyses are no longer adequate. For flow cytometry to meet
the 21st century demands of biotechnology and cell biology, minor
advances in instrumentation will be insufficient. This presentation
demonstrates the effectiveness of a truly next-generation approach to
flow cytometry collection and analysis.
97
SPECTRAL ANALYSIS FLOW CYTOMETER: MODULAR
INCLUSION OF HIGH RESOLUTION SPECTRAL
ANALYSIS
Gregory Goddard1, John Martin1, Mark Naivar1, Peter
Goodwin1, Steven Graves1, Robert Habbersett1, John P.
Nolan1, James Jett1
1
Flow cytometry has established itself as an invaluable tool for general
biomedical research with applications ranging from ligand-receptor
studies, to high throughput screening and genotyping. While
conventional multiparameter flow cytometers have proven highly
successful, there are several types of analytical measurements that would
benefit from a more comprehensive and flexible approach to spectral
analysis including, but certainly not limited to: spectral deconvolution
of overlapping emission spectra, fluorescence resonance energy transfer
measurements, metachromic dye analysis, free versus bound dye
resolution, and Raman spectroscopy. Sensitivity and reproducibility
were investigated with a variety of dispersion methods and image sensor
aspect ratios. Calibration of the prototype spectral analysis flow
cytometer included wavelength characterization and calibration of the
dispersive optics. Benchmarking of the initial system demonstrated a
single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin.
Subsequent improvements to the system resulted in a reduction of the
detection limit to 1100 MESF of FITC. Single particle spectra taken
with our instrument were validated against bulk solution fluorimeter
and conventional flow cytometer measurements with coefficients of
variation of integrated fluorescence intensity of standard fluorescent
microspheres ranging from 2%-5%. It is demonstrated that the flow
spectrometer has sufficient sensitivity and wavelength resolution to detect
single cells and microspheres, including multi-fluorophore or quantumdot labeled microspheres. The capability to use both standard
mathematical deconvolution techniques for data analysis, coupled with
the feasibility of integration with existing flow cytometers, will improve
the accuracy and precision of ratiometric measurements, enable the
116
La Jolla Bioengineering Institute, La Jolla, California; 2Los Alamos
National Laboratory, Bioscience, Los Alamos, New Mexico; 3Los
Alamos National Laboratory, Chemistry, Los Alamos, New Mexico;
4
University of Alberta, Chemistry, Edmonton, Alberta, Canada
Flow cytometry is well-established as a method to analyze the optical
properties of individual particles. Historically these optical properties
have included fluorescence and Raleigh-type elastic light scatter. Raman
scattering is an optical phenomenon that can reveal detailed information
on chemical composition using relatively regions of the optical spectrum.
This property makes Raman spectroscopy of interest for studies of cell
physiology, as well as for microparticle-based and multiplexed assays.
We have developed a flow cytometer capable of detecting and measuring
Raman signals from particles in flow. The instrument features high NA
collection optics coupled to a high throughput spectrograph via an
optical fiber. The Raman spectra is dispersed onto an array of optical
fibers coupled to individual PMTs or onto a CCD array. Nanoparticles
exhibiting surface enhanced Raman scattering (SERS) can be
differentiated on the basis of their spectra, demonstrating their potential
as reporters or as encoding element for particle-based multiplexed
analyses. We are developing this analytical platform to support highly
multiplexed applications in disease diagnostics and drug discovery. The
ability to make Raman spectral measurements of individual particles is
an important new capability for flow based analysis and sorting.
99
FLUORESCENCE SENSITIVITY AND COMPENSATION
IN MULTISPECTRAL FLOW IMAGING
David Basiji1, William Ortyn1, Cathleen Zimmerman1, David
Perry1, David Coder1, Keith Frost1, Brian Hall1, Thaddeus
George1, Richard Esposito1, Richard Bauer1, Philip
Morrissey1
1
The ImageStream system combines advances in CCD technologies with
a novel optical architecture for high sensitivity, multispectral imaging
of cells in flow. Operation of the ImageStream system is similar to flow
cytometry in that data from tens of thousands of cells can be collected
in several minutes. The imagery can be used not only to quantify
fluorescence intensity-based measurements similar to conventional flow
cytometers, but also to measure the distribution of fluorescent signals
and numerous morphologic and absorbance characteristics of each cell.
These capabilities are well suited to study the influence of drug candidates
and disease states on the location and movement of molecules within
and between cells and the associated cell morphology. Effective analysis
requires both high fluorescence sensitivity and accurate spectral
compensation to properly isolate signals within cells. Here we provide
a theoretical treatment of factors governing sensitivity and dynamic
range in multispectral imaging and discuss their dependence on object
size. We describe the compensation process which is conceptually similar
to conventional flow-based compensation, but must be applied at the
individual pixel level and requires the addition of several critical
processing steps to eliminate the generation of artifacts. We develop an
objective statistical metric for comparison of sensitivities between
disparate instruments and show how alternative modes of multispectral
imaging are applied to substantially improve fluorescence sensitivity.
Finally, we show data collected on both the ImageStream multispectral
imaging system and a recent generation 18 bit PMT-based flow cytometer
to compare fluorescence sensitivity by application of the statistical metric.
100
MODULAR, EXPANDABLE, HIGH-SPEED DIGITAL DATA
ACQUISITION SYSTEM DESIGNED TO SUPPORT
MIXED MODE DATA COLLECTION FROM PMTS,
PHOTON-COUNTING APDS, AND HIGH-RESOLUTION
CCD ARRAYS
Mark Naivar1, James Jett2, Jimmy Parson1, Robert
Habbersett1, Steven Graves2, John Martin3, Mark Wilder1,
John P. Nolan4, James Freyer3
1
Resource, Los Alamos, New Mexico; 2Los Alamos National
Laboratory, Bioscience, Los Alamos, New Mexico; 3Los Alamos
National Laboratory, Los Alamos, New Mexico; 4La Jolla
Bioengineering Institute, La Jolla, California
In support of biologically driven goals, we have developed a flexible
and modular data acquisition system for flow cytometry that supports
mixed-mode data collection from a wide range of detector types including
conventional current-mode devices (PMTs, photo-diodes, etc.), singlephoton sensing APDs, as well as high-resolution CCD arrays. Using a
combination of custom acquisition electronics employing FPGAs and a
high speed ADC combined with commercial DSP boards (1 DSP for 4
detectors), the system is scaleable to support high-speed multi-laser
analytical and sorting systems. Although the initial system runs on
Linux, by using Open-Source and cross-platform tools where possible
– such as FireWire and the “QT” GUI development software – the
system can easily be ported to Mac or Windows. To date, the software
has successfully been ported to both platforms with the exception of the
FireWire interface, which should be completed by Jan 2006. In the
signal path, after minimal analog circuitry, a free- running 40MHz 14bit ADC allows us to over-sample all but the shortest signals to give
increased dynamic range. Combined with a DSP to handle the real-time
processing of the raw digitized waveforms, we can generate parameters
unavailable on commercial instruments such as pulse symmetry and
time on each channel with 1 uSec resolution. The system has
demonstrated a large dynamic range (> 4 decades) and the ability to
resolve particles with very low levels of fluorescence. It has been tested
on two very different cytometers: a commercial cytometer with PMTs
and a photodiode that generates pulse widths ~ 40uSec, as well as a
custom cytometer using PMTs and single-photon sensing APDs with
transit times over 1 millisecond. In the operational mode in which the
event-based parameters are extracted by the DSP boards, the system
generates 6 parameters from 32 detectors at up to 10K events/sec, for a
total of 192 parameters. With the pulse-parameter processing code
operating in the FPGAs the throughput is 10 – 20 X higher. This work
was supported by NIH RR020064-01 and NIH RR001315-23.
Cell Physiology 3
101
CORRELATED MULTIPARAMETER FLOW CYTOMETRY
AND MICROSCOPY DEMONSTRATE THAT THE RARE
MOUSE SMALL INTESTINAL EPITHELIAL
PROGENITORS EXPRESSING NEUROGENIN 3 ARE
SLOWLY CYCLING CELLS DERIVED FROM A
BIPOTENTIAL PRECURSOR AND GIVE RISE TO
ENTEROENDOCRINE CELLS
Matthew Bjerknes1, Hazel Cheng2
1
University of Toronto, Medicine, Faculty of Medicine, Toronto,
Ontario, Canada; 2University of Toronto, Medicine, Toronto,
Ontario, Canada
The lining of the small intestine, the epithelium, is organized into
numerous crypts and villi and undergoes continuous renewal. The stem
cells (S) are found among the immature columnar cells in the stem-cell
zone (SCZ), the first 4-5 cell positions of the crypt base. S give rise to
the columnar (C), mucous (M), Paneth (P), and enteroendocrine (E)
cells through a poorly understood process. Neurogenin 3 (Neurog3) is
a transcription factor involved in lineage specification. It is expressed in
rare cells in the crypt. These cells are thought to be progenitors committed
to the E lineage because Neurog3 null mice lack E. However, in transgenic
mice containing floxed lacZ and Cre recombinase under control of a
Neurog3 BAC >10% of M and P cells were lacZ+, indicating that Neurog3
is expressed in a variety of progenitors giving rise various lineages. To
better characterize the role of Neurog3 + progenitors we undertook a
quantitative study. Intestinal epithelium was isolated from proximal
jejunum, stained with antibodies for Neurog3 and Ki-67, then DAPI
and analyzed on a CyFlow ML (Partec GmBH, Germany) flow cytometer
equipped with 488, 532, and 633nm lasers and a mercury arc lamp.
WinList and ModFit (Verity software house, ME) were used for gating
and DNA histogram analysis, respectively. Isolated intact crypts stained
with various antibodies and lectins were examined with a Zeiss
AxioImager Z1 and images recorded with a cooled CCD AxioCam.
Flow cytometry demonstrated that about 0.0018 of epithelial cells are
Neurog3 +. Surprisingly, only 1/3 of Neurog3 + cells are proliferating,
the rest are Ki-67-. We also measured DAPI fluorescence of Neurog3+Ki67+ cells and found that ~19% were in S phase, indicating that Neurog3+
progenitors have a relatively long cell cycle time of about 40 hours (S
phase is about 7-8 hours in the crypt). Direct counts using fluorescence
microscopy confirmed that 0.001-0.002 of epithelial cells are Neurog3+,
1/3 of which are proliferating. The rest are post-mitotic cells in various
stages of E differentiation. Crypts contain on average 1 Neurog3+ cell,
most were located above the SCZ in positions 5-8. Analysis of the cell
types contained in clones derived from single progenitors labeled by
somatic mutagenesis of SWR mice 3 days prior to analysis demonstrated
that Neurog3+ cells are derived from a bipotential progenitor. The various
results are quantitatively consistent with a model in which E are derived
from MixE, a bipotential progenitor whose mitosis yields short-lived
E1 and C1 progenitors committed to E and C lineages, respectively. The
post-mitotic offspring of E1 become Neurog3 in about 1 day and leave
the crypt in about 3 days. We found no evidence of significant
contribution of Neurog3 + cells to other lineages.
102
THE RELATIONSHIP BETWEEN DIFFERENT
NEOPLASTIC CERVICAL LESIONS AND THE
FREQUENCY OF ANEUPLOID CELLS WITH DNA
AMOUNT GREATER THAN 5C
Wang Jian1
1
Canada BC Cancer Agency, Wuhan, Hubei, China
Objective: To determine if there is a relationship between different
grades of neoplastic cervical lesions and the presence and frequency of
aneuploid cells with DNA amount greater than 5c in cytological
preparations. Methods: Cervical samples were taken by a cervix brush.
Cell monolayers were prepared by first washing the brushes in SedFix
and following chemical (DTT) and mechanical treatment of cell
suspension to obtain a uniform single cell suspension, the cells were
deposited onto microscope slides by cyto-centrifugation. Two slides
were prepared from each case: one slide was stained by Papanicolaou
stain for conventional cytology examination, while the other slide was
stained by Feulgen-Thionin method to measure relative of amount of
DNA in cell nuclei using an automated DNA imaging cytometer. The
presence and the percentages of aneuploid cells, those where the cell
nuclei contained DNA amount greater than 5C, were then determined
for each sample. Results: All cases of LSIL and HSIL as called by
conventional cytology and all cases with at least one cell nucleus having
DNA amount greater than 5C (DNA Index>2.5) as determined by
automated DNA image cytometer were sent to colposcopy examination
and biopsy. A total of 496 cases of cervical intraepithelial lesions and
invasive carcinoma were found. The sensitivity of 82% at the specificity
of 74% was calculated for DNA quantitative cytology and sensitivity of
52% at 86% specificity for conventional cytology for combined CIN3
lesions and invasive cancers. The percentages of aneuploid cells having
DNA amount greater than 5c were determined for CIN1, CIN2, and
CIN3 lesions and invasive cancers and these were 0.6±0.04, 1.21±0.16,
2.47±0.38, and 3.12±0.78, respectively. Aneuploid cells were found in
29% of CIN1 cases, in 59% CIN2 cases, 84% of CIN3 cases and in 87%
of invasive cancer cases. Conclusions: There is a clear relationship
between the severity of the neoplastic cervical lesions and the frequency
of aneuploid cells with DNA amount greater than 5c found in cytological
preparations. Therefore the appearance of such aneuploid cells could
be used as a biomarker for detecting high grade CIN lesions and invasive
cancers of uterine cervix.
117
103
COMPARISON OF FLUORESCEIN AND
PHYCOERYTHRIN CONJUGATES FOR QUANTIFYING
CD20 EXPRESSION ON NORMAL AND LEUKEMIC BCELLS
105
APPLICATION OF RATIOMETRIC CELL
ENUMERATION TO THE DEVELOPMENT OF FLOW
CYTOMETRY CELL CITOTOXICITY ASSAYS
Gerald Marti1, Lili Wang2, Fatima Abbasi3, Adolfas
Gaigalas4, Robert F. Vogt5
David Diaz1, Alfredo Prieto1, Hugo Barcenilla1, Jorge
Monserrat1, Luis Chara1, Julio Chevarria1, Miguel Ángel
Sánchez1, Eduardo Reyes1, Melchor Álvarez-Mon2
1
1
United States Food & Drug Administration CBER, Bethesda,
Maryland; 2National Institute of Standards and Technology,
Biochemical Science Division, Gaithersburg, Maryland; 3* CBER
FDA. *Flow and Image Cytometry, Bethesda, Maryland; 4National
Institute for Standards and Technology, Gaithersburg, Maryland;
5
CDC, Division of Laboratory Sciences, Atlanta, Georgia
Quantitative fluorescence calibration has been advocated for the interlaboratory data comparison and the quality control of clinical flow
cytometers for more than a decade. At the present, numerous fluorescence
quantification methods have been developed and instrument calibration
kits have been produced. Due to the lack of follow-up support and
consensus on resolving the differences with the use of these methods
and materials, the quantification efforts have so far a limited clinical
impact. We attempted to quantify CD20 receptor expression on B
lymphocytes as a potential disease biomarker using two different and
yet widely used methods. We measured CD20 expression on normal
and B-CLL B-cells using both FITC and PE conjugates from the same
monoclonal antibody (Mab). As a biological control and calibrator,
CD4 expression on T-cells was also quantified using FITC and PE Mab.
Calibration with QuantiBRITE PE-labeled microspheres and the use of
unimolar PE conjugates provided direct measurements of antibody
bound per cell (ABC) values for CD4 and CD20. Calibration for FITC
conjugates was based on molecules of equivalent soluble fluorochrome
(MESF) as determined by NIST RM 8640 microsphere standards. These
MESF values were then converted to ABC values using the CD4 T-cell
as a biologic calibrator to normalize FITC and PE results for CD20
expression. We demonstrated that CD4 expression on T-cells could be
used as a biological calibrator to quantify CD20-FITC ABC with
reasonable agreement between the two conjugates with two different
fluorochromes. Issues regarding the accuracy of MESF microsphere
calibrators and effective fluorochrome per protein ratios for FITC
conjugates will require additional laboratory studies.
104
TRASTUZUMAB AND PERTUZUMAB DIFFERENTIALLY
AFFECT HER2/NEU OVEREXPRESSING BREAST
CANCER CELLS
Simone Diermeier1, Arabel Vollmann1, Andrea Sassen1,
Ferdinand Hofstaedter1, Gero Brockhoff1
1
University of Regensburg, Institute of Pathology, Regensburg,
Bavaria, Germany
Aim: Her2/neu (c-erbB2) overexpression in breast cancer indicates
eligibility for Trastuzumab therapy. However, because Her2/neu
overexpression alone does not assure responsiveness to this regimen
there is a demand for an optimized Her2/neu based therapy. We evaluated
the mechanism of action of Pertuzumab, a different antibody targeting
Her2/neu, in comparison to Trastuzumab on the cellular level with
respect to Her2/neu dimerization, shedding, EGFR/Her1 and Her2/neu
internalization and cell proliferation in vitro. Methods: BT474 and
SK-BR-3 breast cancer cell lines, both strongly overexpressing Her2/
neu, were treated with Trastuzumab or Pertuzumab. Flow cytometry
was applied to investigate EGFR and Her2/neu internalization, receptorinteraction by FRET and cell proliferation. Shedding of the Her2/neu
extracellular domain was evaluated by Protein Array Technology.
Results: In both SK-BR-3 and BT474 cells Her2/neu homodimerization
was inhibited by Pertuzumab, whilst Her2/neu homodimers seemed to
be stabilized by Trastuzumab treatment. Trastuzumab seemed to inhibit
cell proliferation of BT474 and SK-BR-3 more efficiently than
Pertuzumab. Neither of the antibodies induces EGFR or Her2/neu
internalization. Trastuzumab reduced Her2/neu-shedding in both cell
lines, whereas Pertuzumab diminished it solely in SK-BR-3. Conclusion:
Significant differences in the cellular mechanisms of action of
Trastuzumab and Pertuzumab suggest a potential complementary
antitumour effect if both antibodies are combined in clinical application.
118
Spain; 2University Hospital “Príncipe de Asturias”, Immune system
Diseases and Oncology Service, Alcala de Henares, Madrid, Spain
Traditional cell cytotoxicity assays are based on the labeling of target
cells with 51 chromiun ( 51 Cr). Target cell lysis by cytotoxic cells is
estimated by measuring the gamma radiation emitted by the 51Cr released
from lysed target cells and then percentages of specific lysis of the
target cells can be calculated. Alternative methods based on release of
fluorescent probes have been developed but to discard the excess of
fluorescent probe, intensive washing is required damaging the target
cells and reducing both sensitivity and reproducibility of the assay.
Now, we are studying T cell cytotoxicity against autologous leukemic
B-cell from patients with B-cell chronic lymphocytic leukemia (B-CCL).
These resting target cells incorporate very low quantities of 51 Cr in
comparison with cell lines usually employed as targets in cytotoxicity
assays. We tried to apply fluorometric assays to B-CLL and failed because
B-CLL cells are fragile and suffered necrosis in the procedures used to
wash the fluorescent probes. Therefore, we have to develop a new flow
cytometry method to measure cytotoxicity. In previous works we have
developed methods of cell enumeration of viable and apoptotic cells in
culture and we decided to apply these methods to the enumeration of
cells lysed in cell citotoxicity assays or in the context of drug toxicity.
We designed a flow cytometry assay in which a fixed quantity of target
cells is exposed to different concentrations of apoptogens (drugs or
cytotoxic cells) and we enumerate the number of surviving B-CLL cells
after culture. This method have the advantage that assays can be of
longer duration than the assays based on release of radioactive or
fluorescent labels. We applied this assay to test the efficacy of two in
vitro methodologies to induce cytotoxicity against B-CLL cells. First
we used microbeads as artificial APCs with two Abs against two T cell
antigens CD3 and CD28. This procedure failed to induce cytotoxicity
against B-CLL cells. It even induced helper activity that increased the
survival of leukemic B-CLL cells in vitro. Second we used microbeads
as a bridge between T cells and leukemic cells. The bead has Abs
against a T cell antigen, CD28, and another against a B cell antigen,
CD23. Anti-CD28 and anti-CD23 coated microbeads strongly stimulated
the killing of the leukemic B-cells by autologous cytotoxic T cells. We
have demonstrated the efficacy of these cytotoxic T cells to kill autologous
leukemic B cells in vitro. However when the same assay is realized with
PBMC from healthy controls anti-CD23 and anti-CD28 coated
microbeads do not induce the lysis of non leukemic B cells. This can
mean that in the B-CLL patients there are expanded clones of antileukemic
lymphocytes that are not present in the healthy controls.
106
THE INVOLVEMENT OF
LYSOPHOSPHATIDYLECHOLINE (LPC) IN
LYMPHOCYTE APOPTOSIS IN ATHEROSCLEROSIS
Elena Afrimzon1, Naomi Zurgil1, Yana Shafran1, Mordechai
Deutsch1
1
Biophysical Interdisciplinary Schottenstein Center for Research and
Technology of the Cellome, Physics, Bar Ilan University, RamatGan, Israel
LPC, an intermediate metabolite of phosphatidylcholine, is one of the
major bioactive lipids, which has been identified in atherosclerotic
plaque, and a major component of oxidized LDL (oxLDL), which
plays a key role in atherosclerosis. T-lymphocytes found in early and
late atherosclerotic lesions, actively participate in lesion progression
and rupture. The aim of the present study was to evaluate the apoptotic
effect of LPC on human lymphocytes. The Optical LiveCell™ Array
(Molecular Cytomics Inc.) technology, which enables quantitative
measurements of individual, non-tethered living lymphocytes was
employed to assess multiple functional apoptotic assays followed by
post-fixation studies. Stimulation of activated peripheral blood
lymphocytes, as well as Jurkat-T cell lines, with LPC, triggered apoptosis
in association with an increase in rate of production of reactive oxygen
species (ROS) and an elevation in Bax/Bcl-2 ratio. Real time kinetic of
EGFP-Bid translocation was evident upon treatment of Jurkat-T cells
that were transiently transfected with pd4eGFP-Bid plasmid. Additionally,
LPC induced dose- and time-dependent changes in mitochondrial
membrane potential, concurrently with the increase in ROS production
rate, as measured at the individual cell resolution. In lymphocytes derived
from unstable angina patients, exposure to LPC triggered neither an
apoptotic manifestations nor an increase in ROS generation, and was
associated with a significantly lower ratio of Bax/Bcl-2 expression. This
data suggests that lymphocyte apoptosis induced by LPC may occur via
several intracellular pathways, and may play an important role
progression of atherosclerosis.
Immune Monitoring
107
QUANTITATIVE ANALYSIS OF ANTIGEN-SPECIFIC
ANTIBODY RESPONSES
Henri Van Der Heyde1, William P. Weidanz2, James Burns3,
Irene Gramaglia1, John P. Nolan1
1
La Jolla Bioengineering Institute, La Jolla, California; 2University
of Wisconsin-Madison, Medical Microbiology and Immunology,
Medical School, Madison, Wisconsin; 3Drexel University,
Philadelphia, Pennsylvania
Antibodies play a central role in the response to infection, and assessing
the quantities and affinities of antibodies is fundamental for
understanding the immune response and for developing effective
vaccinations. Most often, antibodies in plasma or serum are assessed in
a qualitative or semi-quantitative manner using ELISA-type methods.
In general, these methods do not allow discrimination of high abundance
low affinity antibodies from low abundance high affinity antibodies.
Moreover, sample volume requirements preclude exhaustive
characterization of antibody types and isotypes. To improve both the
efficiency and the accuracy of antibody characterization, we have
developed as set of methods that use microspheres as solid supports. To
measure the antigen binding activity (a combination of abundance and
affinity) of a plasma sample, antigen-coated beads are used to capture
specific antibodies from plasma, which are then detected using
fluorescence-labeled secondary antibodies specific for antibody types
or sub-types. Calibration of the flow cytometer and measurement of the
fluorophore to protein ration and relative quantum yield of the
fluorescent reporter antibody allow expression of data in terms of
molecules of antibody per microsphere. To deconvolve antibody
abundance and affinity, we estimate the Ab affinity by two methods.
The first titrates soluble antigen in against a single dilution of plasma in
a competition with the microsphere-immobilized antigen. In the second
approach, plasma antigen is captured on to beads using immobilized
type- or sub-type-specific antibodies, and the equilibrium binding of
fluorescence-labeled antigen is measured. These approaches allow the
affinity of the plasma antibodies for antigen to be estimated. We are
using these methods to study the antibody response of mice to
Plasmodium infection in an experimental model of malaria. We find
significant differences in the nature of the antigen-specific antibody
responses to the msp-1 and ama surface proteins during infection.
108
EVALUATION OF ITAG MHC TETRAMERS FOR
PREDICTION OF RECURRENT OR PERSISTENT
CYTOMEGALOVIRUS INFECTION, DISEASE AND
ASSOCIATED TRANSPLANT-RELATED MORBIDITY OR
MORTALITY IN ALLOGENEIC STEM CELL
TRANSPLANT RECIPIENTS: A PROSPECTIVE
MULTICENTER CLINICAL TRIAL
Jan W. Gratama1, Michael Boeckh2, Ryotaro Nakamura3, Rik
A. Brooimans1, Jan J. Cornelissen1, John A. Zaia3, Stephen J.
Forman3, Karl Gaal3, Gail H. Gasior4, Linda A. Sullivan4,
Christopher S. Boyce4, Paula C. Southwick4
1
Erasmus Medical Center, Department of Medical Oncology, Daniel
den Hoed Cancer Center, Rotterdam, Netherlands; 2Fred Hutchinson
Cancer Research Center, Program in Infectious Diseases, Seattle,
Washington; 3City of Hope National Medical Center, Division of
Hematology and Bone Marrow Transplantation, Duarte, California;
4
Beckman Coulter, Inc., Clinical Research, San Diego, California
Study Objective: Cytomegalovirus (CMV) infection is an important
cause of morbidity and mortality in stem cell transplant (SCT) recipients
despite the introduction of routine post-transplant virologic monitoring
and the use of potent antiviral agents. This prospective multicenter
study evaluated the use of tetramers in monitoring CMV-specific T-cell
recovery following allogeneic SCT to predict patients at risk for CMVrelated complications. Methods: Patients were tested every 2 weeks and
monitored for up to 1 year post-transplant. iTAg MHC Tetramers
(Beckman Coulter, San Diego) were used to enumerate CMV-specific
CD8+ T-cells by flow cytometry using a single-platform absolute counting
method. The following tetramers were included: pp50: A*0101
VTEHDTLLY; pp65: A*0201 NLVPMVATV, B*0702 TPRVTGGGAM,
B*3501 IPSINVHHY; IE-1: B*0801 ELRRKMMYM. All patients
underwent weekly surveillance by pp65 antigenemia, DNAemia, or
shell vial culture, with preemptive antiviral therapy. Results: Data were
analyzed for 83 CMV-seropositive recipients with 3 or more tetramer
values. Median follow-up was 9 months (range 2-12). Delayed recovery
of CMV-specific CD8+ T cells (< 7 cells/uL in all blood samples during
the first 65 days post-transplant) predisposes patients to CMV-related
complications. These patients are 2.6 times more likely to develop
recurrent or persistent CMV infection, 4.8 times more likely to develop
CMV disease, 2.4 times more likely to develop fatal complications, and
2.2 times more likely to develop one or more of these outcomes than
patients showing rapid recovery (p=0.01). Rapid recovery (>= 7 cells/
uL in any blood sample during the first 65 days post-transplant) was
associated with protection from CMV-related complications. Interassay
variability was <=8%, and results were available in 3 hours. Conclusion:
CMV tetramer-based immune monitoring, in conjunction with virologic
monitoring, can be an important new tool that permits clinicians to
assess the risk of CMV-related complications and to guide preemptive
therapeutic choices. For high-risk patients with delayed immunologic
recovery by day 65, we suggest that virologic monitoring and preemptive
strategies should be continued beyond 100 days post-transplant;
immunologic monitoring may prove useful in determining when
virologic surveillance can be discontinued. For low-risk patients (>= 7
tetramer-positive cells/uL), studies are needed to determine how long
virologic monitoring should be continued, and whether or not
preemptive therapy may be reduced.
109
MICROBEADS AS ARTIFICIAL APCS AND CELL
BRIDGES FOR T CELL CANCER THERAPY
Alfredo Prieto1, Miguel ÁNGEL SáNchez1, Martin
Villarroel1, David Diaz1, Esperanza Perucha1, Jorge
Monserrat1, Hugo Barcenilla1, Manuel LEONARDO AcuñA1,
Melchor ÁLvarez-Mon2
1
Madrid, Spain; 2University Hospital “Príncipe de Asturias”, Immune
system Diseases and Oncology Service, Alcala de Henares, Madrid,
Spain
Cancer results from genetic alterations within tumor cells that promote
their proliferation and resistance to apoptosis, but also to the failure of
the immune system in the destruction of the tumor cells. Adoptive T cell
therapy is achieving its first irrefutable successes in the reprogramming
of immune responses against tumor cells. These successes are based on
the grafting of anti-tumor T lymphocytes to hosts previously
lymphodepleted by chemotherapy. We selected the B-CLL as a model to
experiment this therapeutic strategy, because is the most common leukemia
and is usually treated with lymphodepletive therapy. Furthermore, we
and other have demonstrated that in these patients there is a reactive
expansion of antileukemic T cells which maintain certain control over
the growth of the leukemic cells. We have developed flow cytometry
methods for the study of in vitro interactions between T cells and
leukemic cells. These methods allow the accurate measurement of the
growth and apoptosis of leukemic cells in coculture with T lymphocytes.
Our laboratory is one of the pioneers in the use of microbeads coated
with monoclonal antibodies (MAbs) in the polyclonal stimulation of T
lymphocytes in vitro . Microbeads coated with Abs combinations can
simultaneously stimulate the T cell antigen receptor and costimulatory
receptors such as CD28 working like artificial antigen presenting cells
that can be used to activate and expand antileukemic T cell clones. We
are developing in vitro methodologies to break the immune tolerance
to the leukemic B-CLL cells and to induce the cytotoxic T cell response
against these tumor cells. The first strategy is to polyclonally stimulate
(with anti-CD3, anti-CD28 and IL-2) T lymphocytes from these patients.
119
A second and very promising strategy was pioneered by our laboratory.
It uses microbeads to bridge leukemic cells and T cells. It uses one
antibody against a leukemic cell antigen and other against costimulatory
antigens of cytotoxic lymphocytes (anti-CD28). These microbeads link
the leukemic cell and the cytotoxic T lymphocyte providing
costimulatory signals for the T cell in the case it recognizes one antigen
presented by the leukemic B cell. The application of methodologies of
cell enumeration in coculture has demonstrated the efficacy of antiCD23 and anti-CD28 coated microbeads in the stimulation of cytotoxic
T cells to kill autologous leukemic B-CLL cells. We are now developing
methods of generation of antileukemic effector T cells in vitro. We have
demonstrated the efficacy of these cytotoxic T cells to kill autologous
leukemic B cells in vitro. We also want to identify and select the T cell
subsets with the highest potential for growth and the strongest killing
activity against leukemic cells.
110
STUDY OF CYTOKINE PRODUCTION BY NAÏVE,
EFFECTOR, MEMORY AND REGULATORY T CELLS BY
8 COLORS MULTIPARAMETRIC FLOW CYTOMETRY
Jorge Monserrat1, Hugo Barcenilla1, Angela Hernández1,
Eduardo Reyes1, Martin Villarroel1, Miguel Ángel Sánchez1,
David Diaz1, Alfredo Prieto1, Manuel Leonardo Acuña1,
Melchor Álvarez-Mon2
1
Madrid, Spain; 2University Hospital “Príncipe de Asturias”, Immune
system Diseases and Oncology Service, Alcala de Henares, Madrid,
Spain
The best phenotypic criteria to define naïve, effector and memory T
lymphocytes are subject of debate. Expression of CD45 isoforms
(CD45RA y el CD45RO) has to be complemented with the concurrent
expression of another markers as CD27, CD62L, CCR7 and CD31 to
distinguish between truly naïve T cells and the different types of antigen
experienced T cells: central memory T cells, non terminated effector
memory T cells and terminated effector memory T cells. Regulatory T
cells are another T cell subset with relevant immunoregulartory functions.
Regulatory T cells have been defined as a subset of memory cells.
However, the existence of naive regulatory T cells has been recently
reported Objetive: By the use of multiparameter flow cyometry in eight
colors we have studied the production by cytokines by the different
subsets of naïve, effector and memory T lymphocytes and also the
production by regulatory T cells. By this method we can study
simultaneously five surface markers and intracellular expression of three
cytokines. Studies of expression of TNFá, IFNã, IL-2, IL-4 and IL-10).
We also studied intracellular expression of FOXP3. Materials and
Methods: We have studied peripheral blood mononuclear cells (PBMC)
from 6 healthy controls. PBMC were cultured for six hours in the
presence or the absence of PMA (50 ng/ml) plus Ionomycin (1 ìg/ml).
Monensin (2 ìM) was added to the cultures to retain the produced
cytokines within the producer cells. We used for surface labeling
antibodies against the antigens: CD3,CD4,CD8,CCR7,CD45RA, CD25
CD27 CD31 and for intracellular labeling antibodies against: TNFá,
IFNã, IL-2, IL-4, IL-10 and FOXP3. Results: We have found significant
differences in the intracellular production of TNFá, IFNã, IL-2 defined
as CD3 +CD8 + /-CD45RA + /-CD27 + /-CCR7 +/-. The percentage of IL-2
producing cells is increased in CD4+ naive T cells but not in CD8+ naive
T cells. The decrease in IL-2 production is correlated with an increase
of IFNã and shift of surface markers indicative of transition to effector
memory cells. This increase is more marked in T CD8 lymphocytes
than in CD4 ones, and is associated to and increase in the expression of
TNFá. We have found that there are naive regulatory T cells that
express FOXP3 at levels lower than that found in classical memory T
cells. Conclusion: The different stages of T cell activation are associated
to different patterns of the production of IL-2, INFã y TNFá. The
existence of a naive Regulatory T cell population is confirmed.
120
111
CD8+ T-CELL DYSFUNCTION IN AIDS: A CELLULAR
DEFECT OR AN ABSENCE OF CD4+ T CELLS HELP?
Patrick J Autissier1, Norman L Letvin1, Igor J Koralnik1,
Joern E Schmitz1
1
Beth Israel Deaconess Medical Center, Medicine, Harvard Medical
School, Boston, Massachusetts
HIV and/or SIV infection destroy CD4+ T lymphocytes and the result is
a loss of immune competence. Loss of immune competence has been
attributed to loss of CD4+ T lymphocytes by direct viral killing and an
indirect impairment of the function of uninfected CD8+ T lymphocytes.
CD8 + T-cells of infected individuals have been shown to lack perforin
and normal indicators of maturation, and down modulate CD3 and
CD28. However, it is possible that these CD8+ T-cells defects could be
due to insufficient help. Ca2 + acts as an intracellular messenger that is
involved in very early steps of cell activation. We evaluated Calciumflux after anti-CD3 triggering in different subsets of CD8+ T lymphocytes
of naïve and chronically SIV-infected rhesus monkeys. Using ionomycin
activation, we did not see any differences in Ca-flux of CD8 + T
lymphocytes of SIV- and SIV + monkeys. However, after anti-CD3
triggering, we saw a Ca-flux defect in total CD8 + T lymphocytes.
Interestingly this defect was associated with the CD4+ T-cell counts. The
chronically SIV-infected monkeys with low CD4 + T-cell counts had a
poor response, while the SIV-infected monkeys with high CD4+ T-cell
counts had a good response. We then evaluated different subsets of
CD8+ T lymphocytes for Ca-flux in infected monkeys with low CD4+ T
cell counts. All subsets of CD8 + T lymphocytes had defective Ca-flux
after CD3 stimulation. These data demonstrate that the SIV-infected
monkeys with low CD4+ T-cell counts and high viral load have a Caflux defect in all subsets of CD8+ T lymphocytes. Since this defect was
correlated to the CD4+ T-cell count of the monkeys, we suggest that this
CD8+ T-cell impairment might be due to an absence of adequate CD4+ Tcell help.
112
T CELLS HOMEOSTASIS IN CHILDREN WITH DOWN’S
SYNDROME
Erika Roat1, Milena Nasi1, Leonarda Troiano1, Nicole
Prada2, Marcello Pinti1, Elisa Nemes1, Enrico Lugli1, Roberta
Ferraresi1, Chiara Giovenzana1, Luigi Ciacci3, Ugo
Consolo3, Fiorella Balli4, Ornella Biagioni5, Mauro
Mariotti5, Andrea Cossarizza1
1
Sciences, Modena, Italy; 2University of Palermo, Dept. of
Biopathology, Palermo, Italy; 3University of Modena and Reggio
Emilia, Dept. of Dentistry, Modena, Italy; 4University of Modena
and Reggio Emilia, Dept. of Pediatrics, Modena, Italy; 5AUSL
Modena, Service for Child Neuropsychiatry, Modena, Italy
Down´s syndrome (DS), the best example of accelerated ageing of the
immune system in humans, is characterized by several defects of the T
cell compartment. In DS children, we investigated T cell homeostasis
by evaluating: 1) the capacity of the thymus to produce and release
newly generated lymphocytes (the so called: “recent thymic emigrants”,
RTE, that express the T cell receptor rearrangement excision circles,
TREC); 2) plasma levels of IL-7, the main cytokine involved in the
differentiation, maturation and homeostasis of T cells; 3) the expression
of its receptor, CD127; 4) extrathymic differentiation of T cells by 9
colour flow cytometry, to identify the main characteristics of virgin and
memory subsets (detected by using CD45RA and CCR7), including the
expression of molecules involved in T cell activation and death (CD38
and CD95 respectively). A 16-parameters CyFlow ML (Partec GmbH,
Muenster, Germany), equipped with a blue solid state laser (488 nm,
200 mW), a UV Mercury lamp HBO (100 long life, 100 W), a red diode
laser (635 nm, 25 mW), a green solid state laser (532 nm, 50 mW) and
a CCD camera was used. Analyses were performed on a total of 31 DS
children (1-11 years old) and 27 age- and sex-matched healthy children
as control. DS children had a significant lower number of RTE and
CD45RA+CCR7 + naïve T cells, while CD45RA-CCR7 + central memory
T cells where more abundant. In particular, CD45RA- CCR7- effector
memory cells augmented in the CD8 + compartment. In DS children but
not in controls a strong correlation was found between age and the
levels of TREC+ cells, while the expression of CD127 was similar. DS
also had significantly higher IL-7 plasma levels. T cell activation status,
as assessed by CD38 expression, was not different between the two
groups. However, we found a remarkable overexpression of the cell
death receptor CD95 in practically all T cell subsets from DS children,
whose overexpression is known to be associated to IL-7 induced T cell
proliferation. The direct measure of thymic output confirms the presence
of a relevant impairment of the thymus, with a reduced production of
RTE. High IL-7 plasma levels, together with the decrease of the naïve/
memory T cell ratio and the increase of CD95 expression, suggest the
presence of a compensatory proliferative mechanism that counteracts a
disorder of lymphocyte compartment, which could be related to the
increased tendency to develop leukaemia/lymphoma typical of DS.
Image Spectral Analysis
113
INCREASING THE LUMINESCENCE OF
LANTHANIDE(III) COMPLEXES
Robert Cary Leif1, Sean Yang2, Alfred Bromm3, Margie C.
Becker4, Lidia M. Vallarino3
1
Newport Instruments, R&D, San Diego, California; 2Phoenix Flow
Systems, Newport Instruments R&D, San Diego, California;
3
Virginia Commonwealth University, Chemistry, Richmond,
Virginia; 4Phoenix Flow Systems, Development & Manufacturing,
San Diego, California
Background: Lanthanide complexes have found a variety of significant
applications in laboratory diagnostics. To achieve the same level of use
in cytometry, the emission intensity of these complexes had to be
increased and improvements were needed in both instrumentation and
software. Methods: The emission intensity of a lanthanide macrocyclic
complex (Quantum Dye®) was increased by performing the
measurements under anhydrous conditions, on solid-state samples
embedded in a mounting medium, and in the presence of a large excess
of molecules and/or ion complexes capable of functioning as energytransfer antennas. This increase in luminescence intensity appears to be
the result of the fluorescence energy transfer enhanced luminescence,
FRETEL, effect. Since a large excess of the luminescence-enhancing
antenna species was not complexed to a lanthanide(III) ion, the energytransfer process may have involved singlet-state rather than triplet-state
emitters, thus significantly increasing the number of species available
for energy transfer. For time-gated images, the excitation light source
was a UV LED with a long excitation period and a short time to turnoff.
A CCD camera was used to produce a summation of the time-gated
images. Special software was written to analyze and present these images
in a pseudo-absorbance mode. Results: New chemical protocols that
enhance luminescence intensity were combined with improved
instrumentation and special software to produce images of apoptotic
and S phase cells stained with the europium Quantum Dye conjugate of
anti-5BrdU and DAPI. The time-gated images of the europium emission
were free from contamination by DAPI emission and were presented on
a white background consistent with common medical usage.
114
LIPID INTERACTION AND LOCALIZATION WITH NILE
RED BY FRET MULTIPHOTON CONFOCAL SPECTRAL
IMAGING
Edmond Kahn1, Vejux Anne2, Dominique Dumas3, Frouin
Frederique1, Gerard Lizard2
1
3
INSERM U678, Paris, France; 2INSERM U498, Dijon, France;
University of Nancy, Vandoeuvre-Les-Nancy, France
Among oxysterols, 7-ketocholesterol (7KC) is able to trigger apoptosis
on various cell types (including those of the vascular wall). Indeed,
7KC is frequently detected at high levels in atherosclerotic plaques, and
apoptotic cells are often observed in atherosclerotic lesions. Human
promonocytic U937 leukemia cells were therefore cultured in the absence
or presence of 7KC and to determine the effects of 7KC on lipid content,
U937 cells were stained with Nile Red (NR). NR emits a yellow
fluorescence in the presence of neutral lipids (cholesterol esters,
triacylglycerols) and an orange-red fluorescence in the presence of
polar lipids (phospholipids, sphingomyelin) when it is excited by a blue
light. Thus, when the blue emission of the ultraviolet (UV) excited 7KC
as donor is accepted, excitation of NR occurs when co-localization of
7KC and NR exists. This work investigates the effect of 7KC on cellular
lipid content and the interaction of 7KC with NR. Via two-photon
excitation confocal laser scanning microscopy (CLSM), UV-like
fluorescence resonance energy transfer (FRET) excitation of NR on
U937 cells is performed. Untreated and 7KC-treated U937 cells are
stained with NR. 3D sequences of images are obtained by spectral analysis
in a two-photon excitation CLSM and analyzed by the factor analysis of
medical image sequences (FAMIS) algorithm, which provides factor
curves and images. In this method factor curves and images are the
result of FAMIS image processing which uses physical properties of
fluorochromes (i.e.: emission spectra) to identify spectral patterns of
fluorochromes as factor curves and restore the corresponding images as
factor images. In FRET analysis, preparations are screened at selected
wavelengths (i.e.: 760nm) to avoid emission of NR in the absence of
7KC. During 7KC-induced cell death, CLSM reveals a modification of
the cellular lipid content. Factor images show FRET occurrence, and
subsequent co-localization of 7KC and NR. Our investigation establishes
the utility of two-photon excitation CLSM to assess co-localization of
7KC with NR by FRET and to identify and distinguish polar and neutral
lipids stained by NR which accumulate under the effect of 7KC.
115
NEXT-GENERATION CYTOMETRY: SPECTRAL
FINGERPRINTING
Bartlomiej Rajwa1, Valeri Patsekin2, J. Paul Robinson3
1
West Lafayette, Indiana; 2Purdue University, W. Lafayette, Indiana;
3
Engineering, West Lafayette, Indiana
This presentation will offer a proposal that the future of cytometry may
well be to redefine the fundamental basis for flow cytometry design by
integration of principles of chemical analysis and image processing.
The process is based on flow cytometry combined with spectral
fingerprint analysis. Such a system would consist of a specially designed
high-speed multichannel detector, and spectral analysis & classification
software. By using spectral analysis technology as opposed to a
fluorescence intensity profile analysis we can implement capabilities
for automated classification. The opportunities for next-generation
cytometry will expand current screening devices used in cytomics &
high-speed classification analyzers with intelligent diagnostic capabilities
useful for clinical pathology & rapid identification of unknown samples
for example. This is a paradigm shift in the design of cytometers. The
new paradigm establishes spectral fingerprint analysis as a core method
of screening and classification, in contrast to current intensity-based
techniques. These well-established screening methods currently used in
diagnostic instruments rely on organic fluorochromes & emission banddetection systems. They are powerful, but by nature are probably
restricted to a dozen or so simultaneous fluorescent probes, have
complicated and numerous optics as well as restricted spectral bandwidths,
& must also deal with the very complex issue of compensation. This
method of signal acquisition is not well suited for spectral fingerprint
detection, where sensitivity and versatility may be sacrificed for the
benefit of higher spectral resolution. While there are significant issues
to overcome, we believe that our approach, if successful will create a
series of fundamental changes in cytometry approaches. First, by using
spectral analysis we can utilize a vast resource of capabilities from the
remote sensing and chemistry communities for analysis. Second, the
advances in computers and electronics affords an opportunity for smaller
quite sophisticated instruments at a relatively low cost. Third, the
introduction of advanced analytical approaches to cytometry analysis
will open many opportunities previously too difficult to implement.
There are many issues that must be successfully resolved in order for
such a technology to be successful; design of an electronic system of
detection with a reasonable signal strength to allow adequate data
collection via new optical designs, adaptation of staining technologies
to become useful for biologically compatible spectral coding and design
of quite advanced integrated software package for rapid analysis of
spectral data. We will demonstrate the powerful nature of the above
approach using biological and non-biological examples.
121
116
MULTISPECTRAL IMAGING METHODS FOR MULTILABEL MICROSCOPY
Richard Levenson1, James Mansfield1
1
CRI, Woburn, Massachusetts
Detection of multiple molecular species simultaneously in microscopy
is becoming increasingly important, and both multilabel chromogenic
and fluorescence methods have been developed. In fluorescence
microscopy, detectability and accuracy of quantitation can be limited
by the presence of autofluorescence, particularly in formalin-fixed tissue
samples. In addition, visualization of co-localized events in fluorescence
can be difficult when one signal is much brighter than another.
Multispectral imaging (MSI), in which the emitted signals are spectrally
resolved, can be used to overcome these difficulties. Implementation of
MSI in the context of laser-scanning confocal microscopy is now
popular. Other techniques, such fluorescence lifetime measurements,
can also resolve multiple signals, but these require sophisticated gated
sources and/or detectors.
Alternatively, high-quality MSI can be
achieved using electronically tunable band-pass-based instruments which
have the advantages of optical flexibility (no confocal microscope
required), better light budget, simplicity and considerably lower cost.
Liquid-crystal-tunable filter (LCTF)-based MSI is shown to be useful
for multicolor FISH, for resolving multiple species of GFP, for high
levels of multiplexing with quantum-dot reagents, and in many cases
for removing ubiquitous autofluorescence signals. In particular, MSI
can be critical for use with clinical pathology specimens, in which
formalin fixation greatly increases background fluorescence.
Clinical
pathologists prefer to use brightfield microscopy over fluorescence,
achieving
molecular
imaging
through
chromogenic
immunohistochemistry. However, multiplexing is harder to achieve,
since overlapping signals can rarely be resolved visually or with
conventional imaging techniques. MSI, on the other hand, allows the
separation and quantitation of up to 3-4 overlapping signals deposited
as part of immunohistochemical or in-situ hybridization procedures.
The unmixed individual component images can be segmented and
quantitated using simple image analysis methods which are
straightforward to automate. We will show examples including
simultaneous brightfield detection of double nuclear markers and a
counter-stain.
Software tools for unmixing, and quantitating multilabel samples are extremely important. The ability to accurately determine
the spectral qualities of dyes in situ proves to be particularly valuable.
Appropriately constrained linear unmixing algorithms and novel
automated tools have recently been developed to provide simple,
accurate analysis procedures. In addition, methods for the conversion
of unmixed images to information of relevance to pathologists are
presented.
117
MULTISPECTRAL FLUORESCENCE IMAGING OF
SMALL ANIMALS: DETECTION AND ANALYSIS
METHODS USING ACOUSTO-OPTIC FILTERING
Silas J. Leavesley1, Valery P. Patsekin2, Wamiq Ahmed3,
Bartlomiej Rajwa4, J. Paul Robinson5
1
Purdue University, Biomedical Engineering, West Lafayette,
Indiana; 2Purdue University, Bindley Bioscience Center, W.
Lafayette, Indiana; 3Purdue University, Electrical and Computer
Engineering, Schools of Engineering, West Lafayette, Indiana;
4
& Biomedical Engineering, West Lafayette, Indiana
Small animal fluorescence imagers, although a relatively new
development, have become a widely available technology in many
research laboratories and veterinary institutes. The ability to monitor
tumor and disease progression, protein expression, and cellular
proliferation in vivo are incredibly powerful techniques, and have
already had an impact in many fields of research. Currently, the main
limitations of fluorescence imaging in vivo are imaging depth (due to
photon absorption and scattering), background noise (often created by
autofluorescence), and excitation and emission limitations of some
fluorescent dyes. Multispectral imaging techniques help to decrease
the limitations of small animal fluorescence imaging by selecting multiple
spectral bands at which to acquire images. By applying filtering
algorithms, it is possible to remove noise from an image set, improving
the SNR and possibly the imaging depth. However, many multispectral
122
imaging systems operate very slowly, because the filtering removes a
large portion of the available emission photons and because multiple
images must be taken. In addition, there is also an inherit time required
to switch between different filtering wavelengths. This slow speed can
create problems when performing time-based kinetics studies and can
increase the contribution of motion artifacts.
This research describes
the application of acousto-optic filtering technology for the acquisition
of multispectral data sets for visualization within a reasonable time
frame. It also describes the application of various methods for easy
visualization and analysis of multispectral data sets for the purpose of
classification.
118
CONFOCAL MICROSCOPY SYSTEM
PERFORMANCE:SPECTROSCOPY
Robert MARTIN Zucker1, Jeremy Lerner2
1
U.S E.P.A. Research Triangle Park, Research Triangle Park, North
Carolina; 2LightForm Inc, Hillsborough, New Jersey
The confocal laser-scanning microscope (CLSM) has enormous potential
in many biological fields. The goal of a CLSM is to acquire and quantify
fluorescence and in some instruments acquire spectral characterization
of emitted signals. The accuracy of these measurements demands that
the system be in correct alignment with stable laser power and spectral
registration. This evaluation can not be made using a histological slide
to create a “pretty picture” which appears to be the most common
method to check the performance of a CLSM system. We have developed
tests to replace this subjective approach with objective measurements of
field illumination, lens function, total laser power, laser stability, dichroic
reflectance, axial resolution, galvanometer scanning stability, overall
machine stability, and system noise (1-2). We have developed additional
tests to measure spectral performance to serve as guidelines for
investigators to assess both the performance of their instruments as well
as the quality of their data. (3, 4) The spectral characterization test is
well suited to all wavelength dispersive CLSM systems including the
Leica SP, Zeiss Meta, Olympus FV 1000, and Nikon C1 confocal
microscopes. We used a multi-ion discharge lamp (MIDL) (LightForm,
Inc., Hillsborough NJ) containing mercury ions and inorganic
fluorophores as a light source because it emits stable reference peaks
between 400 and 650 nm. The spectral features of the MIDL lamp
include peaks at 436, 485, 545 586 and 611nm. The signature of peaks
and valleys is a measure of the accurate representation of wavelengths
of light by the CLSM. The lamp is simply positioned on the microscope
stage above the objective lens (3). The characteristics of the acquired
spectrum enable us to measure spectral sensitivity, contrast, wavelength
ratios and spectral resolution. Since the wavelength position of the
emission peaks is reliable and reproducible, it is possible to compare the
performance of all three PMT´s in one Leica system and the difference
in spectral response between similar systems in different laboratories.
Confocal microscope misalignment and instability in spectral imaging
systems can be assessed using this MIDL reference standard (4).
References 1. Zucker RM and Price OT: Cytometry 44:273-294 2001 2.
Zucker RM and Price OT: Cytometry 44:295-308 2001 3. Lerner JL
Zucker RM: Cytometry 62A:8-34 2004 4. Zucker RM Lerner JL:
Microscopy Research and Technique: 68:5 307-319 2005 This abstract
of a proposed presentation does not necessarily reflect EPA policy
MIDL spectral pattern-Leica Lambda Scan
Biotechnology
119
THE USE OF MULTI-PARAMETER FLOW CYTOMETRY
FOR THE CHARACTERISATION AND MONITORING OF
INSECT CELL-BACULOVIRUS FERMENTATIONS IN A
MECHANICALLY-AGITATED BIOREACTOR
Christopher J. Hewitt1, Bojan Isailovic1, Ryan Hicks2, Ian
Taylor2
1
University of Birmingham, Chemical Engineering, Engineering,
Edgbaston, , United Kingdom; 2AstraZeneca, Macclesfield, , United
Kingdom
Bacteria and mammalian cells have been traditionally used as hosts for
commercial recombinant protein production. In recent years, the insect
cell-baculovirus system has emerged as a potentially attractive
recombinant protein expression vehicle. This route is attractive because
baculovirus-infected insect cells are able to perform post-translational
modifications whilst accommodating very abundant expression of
recombinant protein. Although flow cytometry has been used widely
for the analysis of mammalian and microbial cell physiology and
morphology, there is very little information on applications of this
powerful and highly efficient technique in insect cell culture. Here we
have compared cell ratiometric counts and the viability of Sf-21 cell
cultures using a flow cytometer to those determined by more traditional
methods using a haemocytometer and the trypan-blue exclusion dye.
There was good agreement between the two counting methods but the
former technique proved to be a more reliable and statistically robust
viability indicator (stains used: propidium iodide and calcein AM). Flow
cytometry has also been used to monitor various parameters during
fermentations of Sf-21 cultures infected with the recombinant
Autographa californica Nuclear Polyhedrosis Virus (AcNPV). This
recombinant baculovirus contains the inserted nucleic acid sequence
amFP486 coding for AM-Cyan coral protein, which emits green
fluorescence when excited at 488nm. A good correlation has been
obtained between parameters such as mean green fluorescence and
AmCyan positively stained cells linked to cell viability. Additionally,
DNA content, cell size and granularity have proven to be good indicators
of baculovirus infection. Additionally, we have simplified and optimised
methods of cell treatment for at-line real-time flow cytometric analysis
of these potentially important processes.
120
DETERMINATION OF FLUORESCENCE IN SITU
HYBRIDIZATION POSITIVE EVENTS WITH AN
IMAGING FLOW CYTOMETER – FISH IN FLOW
Luchuan Liang1, Philip Morrissey1, Brian Hall1, Thaddeus
George1, David Basiji1, William Ortyn1, Keith Frost1,
Cathleen Zimmerman1, Richard Esposito1, Richard Bauer1,
David J Perry1
1
Assessment of FISH spots in cells has traditionally been performed by
manual assessment via microscopy. This has made assessment of large
populations of events or identification of rare populations of abnormal
cells, a time consuming and tedious event. To some extent slide based
imaging cytometers have facilitated analysis, but these are not optimal
for analysis of cells naturally in suspension, e.g., blood cells. In one
example from the scientific literature, the determination of the degree
of aneuploidy in sperm cells is performed via FISH for chromosomes
X, Y and 8 in experimental animals exposed to potential aneugens. In
order to achieve statistical significance in these studies, 10,000 sperm
must be scored manually. Published studies have reported a 2-fold
variation between laboratories in the evaluation of the same sample.
Thus, this field can benefit significantly from rapid image acquisition
and quantitative digital analysis and classification of the anueploid events.
The ImageStream imaging cytometer acquires 6 channels of imagery
simultaneously from cells in flow including bright field, dark field and
4 channels of fluorescent imagery at acquisition speeds of 300 cells per
second. A prototype ImageStream with an additional 375 nm uv laser in
addition to the 488 nm laser was used in these studies. Protocols were
developed to perform in situ hybridization with cell in suspension both
with somatic cell lines and primary cells using probes labeled with a uv
excitable dye as well as the standard 488 nm excitable dyes such as
spectrum green and Cy3. Cell lines and primary somatic cells were
subjected to in situ hybridization with 3 differentially labeled probes
for chromosome enumeration while maintaining them in suspension.
Imagery was then acquired on the ImageStream and analyzed with a
spot counting algorithm. Results on the degree of aneuploidy in cell
lines and primary somatic cells will be presented and compared to
results obtained with the established slide based method.
121
BEYOND ‘ONE-IN-A-BILLION´: RARE CELL SORTING
IN DIAGNOSTICS AND THERAPEUTIC DEVELOPMENT
Patrick S. Daugherty1
1
University of California, Santa Barbara, Chemical Engineering,
Engineering, Santa Barbara, California
Quantitative, high-throughput cell sorting instrumentation is catalyzing
discoveries in proteomics, protein engineering, marker discovery, and
therapeutic development. We recently developed a family of
methodologies that harness rare cell sorting to enable screening of cellbased molecular libraries. Specifically, libraries containing as many as
50 billion members have been constructed and screened to identify
protein, virus, and cell binding ligands, map intracellular protein-protein
interactions, and to discover enzyme substrates. Finally, I will describe
how coupling of these new library methodologies with disposable, chipbased cell sorters offers exciting new prospects in the development of
advanced molecular diagnostics and the engineering of next generation
therapeutics.
122
A MULTIPLEX FUNCTIONAL SCREEN OF PHAGE
DISPLAY LIBRARIES USING FLOW CYTOMETRY
Loretta Yang1, John P. Nolan1
1
La Jolla Bioengineering Institute, La Jolla, California
Phage display, a method of selecting functional peptides or proteins,
can be greatly complemented by flow cytometry. Specific binding clones
within a phage library are traditionally identified by iterative rounds of
panning against a target followed by phage DNA sequencing until a
consensus is obtained. These phage, or the molecules that they display,
are then further analyzed to assess their function. As an alternative to
phage ELISA, phage can be displayed on microspheres to allow for
convenient and rapid analysis by flow cytometry. The specificity and
avidity of the interaction of phage-displayed ligands for the target can
be directly determined. We screened three commercial phage-displaying
peptide libraries against a target protein, the cholera toxin B subunit
(CTB), and clones selected from each library (30 total) were sequenced
in the final rounds of panning. Screening by sequence convergence, a
total of 16 clones were identified and analyzed in detail. To characterize
these clones, we captured phage onto microspheres bearing immobilized
anti-phage antibodies. The captured phage were then incubated with
fluorescein-labeled CTB and the microsphere fluorescence measured
by flow cytometry. We found that 50% (8/16) of the selected phage
showed detectable binding activity, and three showed strong binding at
low (20 nM) CTB. To extend this flow cytometric approach to the
primary screening of clones, we devised a multiplexed target binding
assay using fluorescence-encoded microspheres. Anti-phage antibodies
were conjugated to ten populations of beads that exhibited discrete
intensities of red fluorescence. An aliquot of each bead population was
used to capture phage from a crude culture supernatant. The phage
coated beads were then washed, pooled, incubated with fluorescent
CTB, and their fluorescence analyzed by flow cytometry. This
multiplexed screening approach discriminated among known targetbinding and non-binding phage with 100% accuracy. When used to
screen 144 uncharacterized clones, this approach detected 42 hits, of
which ten were new clones not detected in the previous sequence-based
screening. Upon further characterization, two of the ten new clones had
exhibited strong binding (avidity better than 31 nM). In summary,
function-based screening using microsphere arrays and flow cytometry
is significantly more efficient at finding unique strong binding clones
than traditional sequence-based screening. Supported by NIH EB03824.
123
123
YEAST SURFACE DISPLAY SYSTEM FOR SCREENING
OPTIMAL SUBSTRATE FOR ANTHRAX LETHAL
FACTOR
Weon Bae1, Jian Hong Zhou2, Steven Graves1
1
Los Alamos National Laboratory, Bioscience, Los Alamos, New
Mexico; 2Los Alamos National Laboratory, Los Alamos, New
Mexico
Proteases regulate many diverse biological pathways and are also
common components of many bacterial toxins targeting cell signaling
pathways. Consequently they become attractive targets for
pharmaceuticals and detection assays. However, the study of proteases
and development of inhibitors for toxin proteases does not follow a
single established methodology. Most protease assays and development
of inhibitor screens are specifically designed for the protease of study
and cover a variety of methodologies. In order to develop a highthroughput and generally applicable method for investigating protease/
substrate interaction, we developed a flow cytometry-based assay system
combined with cell surface display. We constructed both a bacterial and
a yeast surface display systems, which express a fluorescing reporter
protein fused to either a short cleavage site or a full length substrate of
the Lethal Factor metalloprotease (LF) of Bacillus anthracis. Interestingly,
a full length of substrate was shown to be superior to a short cleavage
sequence only. Because most of the screening methods use a library of
small peptides, our results Yeast cells expressing different substrates
were incubated with LF to determine the efficiency of cleavage. Cells
expressing all natural substrates, mitogen activated protein kinase kinase
(MAPKK) family, showed the decreasing fluorescence signal after
treatment with LF, whereas the fluorescence signals of the cells expressing
non-related proteins were not changed after the treatment. Interestingly,
cells expressing the consensus sequence of LF substrate were turned out
to be the most efficient substrate in both kinetics and the completeness
of cleavage. We are currently constructing a mutation library of the six
conserved residues to screen them to identify better substrate for the LF.
These studies will elucidate the roles of specific residues within the
substrate cleavage site and how the protease achieves substrate specificity.
This information will be used to evaluate isolated optimal substrates for
use as lead compounds for pharmaceutical development for treatment
of anthrax and as reporter molecules for B. anthracis detection assays.
124
MORE ACCURATE GENETIC DIAGNOSIS BY SINGLE
COPY PROBE QUANTITATIVE MICROSPHERE
HYBRIDIZATION
Heather Newkirk1, Peter K. Rogan2, Mauricio Miralles1, Joan
H.M. Knoll1
1
Children’s Mercy Hospital, Laboratory of Genomic Disorders,
Kansas City, Missouri; 2Children’s Mercy Hospital, Laboratory of
Human Molecular Genetics, Kansas City, Missouri
We developed a novel quantitative microsphere suspension hybridization
(QMH) assay for determination of genomic copy number by flow
cytometry. Single copy (sc) genomic sequences were conjugated to
spectrally-distinct polystyrene microspheres. Conjugated probes were
used in multiplex hybridization to detect homologous sequences in
labeled target genomic DNA extracted from fixed cell pellets from
cytogenetic studies. Prior amplification of target DNA was not required
because sc probes provide adequate specificity and sensitivity for accurate
copy number determination. Hybridization was detected with
phycoerythrin-labeled streptavidin and analyzed by flow cytometry.
Copy number differences were distinguished by comparing mean
fluorescence intensities (MFI) of test probes with a disomic reference
probe (HOXB1) of patient DNA samples and abnormal cell lines. The
MFIs of the ABL1 probes were reduced to 0.59 ± 0.02 fold in 3 different
chromosome 9 deletion patients and increased 1.42 ± 0.01 fold in 3
trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate
copy number increases in 5 patients with Charcot-Marie-Tooth Type 1a
disease and chromosome 17p12 duplications. The assay can therefore
distinguish a single allele and three alleles from a biallelic reference
sequence, regardless of chromosomal context. We also demonstrated
that QMH using sc probes to be more accurate than existing genomic
and expression microarray technologies. We also demonstrate that QMH
measurements using sc probes are more accurate than existing genomic
and expression microarray technologies. sc probes circumvent the
requirement to suppress crosshybridization of repetitive sequences in
124
probes and genomic templates (with Cot-1 DNA). We demonstrate that
adventitious association between sequences in C o t-1 and the probe can
distort quantification of probe hybridization to desired genomic targets.
QMH effectively combines the high throughput advantages of flow
cytometry with the precision of sc probe technology to create a robust
microsphere-based assay for the direct assessment of genomic copy
number.
Apoptosis
125
HYPOXIN-INDUCED MEMBRANE-BOUND APOPTOTIC
DNA PARTICLES: POTENTIAL MECHANISM OF FETAL
DNA IN MATERNAL PLASMA
Aaron Orozco1, Cassandra Horne1, Edwina Jane Popek2, Joe
Leigh Simpson3, Farideh Z. Bischoff4, Dorothy E Lewis5
1
Baylor College of Medicine, Immunology, Houston, Texas; 2Baylor
College of Medicine, Pathology, Houston, Texas; 3Baylor College of
Medicine, Obstetrics and Gynecology, Houston, Texas; 4Baylor
College of Medicine, Obstetrics and Gynecology, Medicine,
Houston, Texas; 5Baylor College of Medicine, Immunology,
Medicine, Houston, Texas
Fetal DNA, found in the plasma of pregnant women, is being studied as
a way to diagnose genetic abnormalities using specific primers and PCR
amplification. Though the underlying mechanism giving rise to this
DNA in plasma remains unclear, our hypothesis, based on the stability
of such DNA, is that the DNA may be contained in apoptotic bodies
(ApoBodies) created from dying cells. Trophoblast apoptosis is essential
for normal placental development as a consequence of proliferation,
differentiation, and migration of these cells during pregnancy. Through
flow cytometric sorting and real-time PCR, we found that Acridine
Orange (AO) stained, high light scatter particles are resistant to DNase
treatment, disrupted by SDS, and contain fetal DNA. Because the placenta
continuously remodels in an hypoxic environment, our hypothesis is
that fetal DNA in maternal plasma comes from hypoxia-induced dying
trophoblasts, which circulate predominately in the form of ApoBodies.
We developed a model culture system for characterization of ApoBodies
derived from JEG-3 cells, an extravillous trophoblastic cell line,
undergoing various methods of cell death caused by hypoxia, etoposide,
and heat stress. Using conditions of similar propidium iodide (PI) uptake,
suggesting comparable levels of death, both hypoxia and etoposideinduced ApoBodies increase by 48 hours (up to about 7 bodies per
cell), whereas, levels of heat induced particles remain fairly constant
over time (at 1 body per cell). Untreated cells produce about 1 body per
5 cells, as measured by quantitative flow analysis using fluorescent
beads as a standard. Thus, hypoxia, which is likely responsible for
trophoblast cell death in vivo, produces membrane-bound (PKH-26
stained) ApoBodies containing DNA (pico green stained) with
characteristic high light scattering properties when placed into plasma.
Our results show that these in vitro generated ApoBodies are similar to
the membrane-bound particles containing fetal DNA we previously
found in maternal plasma. These experiments offer a quantitative flow
cytometric based way to characterize ApoBodies in both normal and
abnormal pregnancies. KEYWORDS: Apoptotic Bodies; Fetal DNA
126
A NOVEL FLOW CYTOMETRY BASED METHOD TO
DETERMINE THE VIABILITY OF BETA CELLS USING
THE ZINC-BINDING DYE FLUOZIN-3 AND THE
MITOCHONDRIAL MEMBRANE POTENTIAL
INDICATOR TMRE
Sundararajan Jayaraman1
1
University of Illinois at Chicago, Surgery, College of Medicine,
Chicago, Illinois
Transplantation of islets isolated from cadaveric donor pancreata is a
current option for the treatment of type 1 diabetes. However, this
promising strategy is severely limited by a number of factors including
the inability to unambiguously determine the viability and function of
insulin-producing beta cells prior to transplantation. Therefore, our
goal was to develop a simple and rapid flow cytometry based assay to
detect live beta cells. We have recently shown that the potentiometric
dye TMRE (tetramethylrhodamineethylester) is superior to other
mitochondria-specific dyes for the determination of viability in insulin-
producing NIT-1 cells (J. Immunol. Methods, 2005, in press). Since the
integrity of the inner mitochondrial membrane is crucial for energydependent processes including insulin secretion, TMRE retention also
reflects the beta cell function. Identification of beta cells was based on
the binding of FluoZin-3 which has higher affinity to zinc than other
zinc-binding dyes. Moreover, unlike other zinc-binding dyes, FluoZin3 is resistant to chemical fixation that is essential for the simultaneous
analysis of various intracellular proteins including insulin in beta cells.
Since beta cells contain higher concentrations of free zinc than most
other cell types, they are expected to bind FluoZin-3 more avidly than
other cells. Simultaneous staining of beta cells with TMRE and FluoZin3 facilitated unambiguous discrimination between live and dead beta
cells. Flow cytometric analysis of intracellular activated caspases
(Cytometry, 2003; 56A: 104), intracellular acidification and
phosphatidylserine externalization validated that only viable beta cells
stain with FluoZin-3 and retain TMRE. Confocal imaging revealed the
co-localization of FluoZin-3 and the mitochondria-specific dye H2CMX-Ros to mitochondria in live beta cells, indicating that free chelatable
zinc is compartmentalized in mitochondria in beta cells. Using this
method, we were able to determine the frequency of beta cell death
under varying experimental conditions. The combined use of the
potentiometric dye TMRE and the zinc-binding dye FluoZin-3 may
prove to be useful for rapid and unambiguous estimation of live and
functional beta cells in islet preparations prior to transplantation into
type 1 diabetes patients.
127
RELATIONSHIP BETWEEN MITOCHONDRIAL
DEPOLARIZATION AND OTHER APOPTOTIC EVENTS
Sundararajan Jayaraman1, Jesus Exposito1, Carlos Salgado1
1
University of Illinois at Chicago, Surgery, College of Medicine,
Chicago, Illinois
Dissipation of mitochondrial transmembrane potential is considered as
the critical point during T cell death but its relationship to activation of
caspases and other apoptotic events has not been fully elucidated.
Apoptosis induction by anti-Fas antibody treatment was accompanied
by rapid mitochondrial depolarization in Jurkat cells as assessed by the
retention of TMRE using flow cytometry. Activation of caspases occurred
concurrently with inner mitochondrial membrane depolarization as
assessed by flow cytometry. Mitochondrial depolarization occurred in
the absence of enhanced oxidative stress and was accompanied by cell
volume shrinkage, intracellular acidification, and phosphatidylserine
externalization. Pretreatment with caspase-6 or caspase-8 inhibitor
prevented Fas-mediated proteolytic cleavage of caspase-2, caspase-8,
caspase-9, BID and PARP. However, inhibition of caspase-6 or caspase8 prevented mitochondrial depolarization, intracellular acidification and
apoptosis only partially. On the other hand, the pancaspase inhibitor ZVAD-FMK fully prevented proteolysis of caspases, BID and PARP, and
blocked all of the cellular manifestations of apoptosis. These data are
consistent with the contention that multiple caspases are involved in
exerting damage to the mitochondria during Fas-mediated apoptosis.
Our data also suggest that although caspases link mitochondrial
depolarization and other apoptotic events, these events appear to be
differentially regulated.
128
FLOW CYTOMETRY LEADS A SHIFT OF PARADIGM IN
THE ETHIOPATHOGENIC ROLE OF T LYMPHOCYTE
APOPTOSIS IN MULTIPLE SCLEROSIS
Hugo Barcenilla1, Alfredo Prieto1, David Diaz1, Jorge
Monserrat1, Manuel LEONARDO AcuñA1, Antonio GarcíAMerino2, Melchor ÁLvarez-Mon3
1
Alcala University, Immune System Diseases and Oncology
Laboratory, CNB-CSIC R&D Associated unit, Alcala de Henares,
Madrid, Spain; 2Clínica Puerta de Hierro, Servicio de Neurología,
Madrid, , Spain; 3University Hospital “Príncipe de Asturias”,
Immune system Diseases and Oncology Service, Alcala de Henares,
Madrid, Spain
Based on the assumption that these differences in apoptosis reflected a
defective induction of apoptosis of T lymphocytes from these patients,
a general defect in regulation of T lymphocyte apoptosis was proposed
as the cause of increased life span of auto reactive T cells and autoreactive
pathology in MS patients. However there are not well performed studies
on the apoptosis of the rest of T lymphocytes from MS patients.
Methods: To ascertain if the apoptosis of peripheral lymphocytes of
MS patients was decreased we performed studies of determination of
apoptosis in individual cells by four independent methods (annexin-V,
Low molecular weight extraction of apoptotic cells, TUNEL and
activation of caspases 3, 8 and 9 by fluorescent inhibitors of caspases)
in a group of 48 MS patients. Results: we found that PBL from MS
patients showed impressive increases in the incidence of spontaneous
apoptosis in the PB T cell populations (CD3, CD4, CD8, CD4CD45RA,
CD4CD45RO, CD8CD45RA, and CD8CD45RO) but not in B cells. More
than half of MS patients showed spontaneous apoptosis values above
the range of values found in the healthy controls. We also found that
the increase in apoptosis in all these populations was of greater magnitude
in the patients suffering exacerbations of the disease and also in those
that have suffered a higher frequency of relapses in the two years previous
to the study. We also demonstrated that IFNâ treatment decreased the
spontaneous apoptosis in those patients in which the treatment was
effective. The patients who despite treatment suffered relapses showed
strong increases in the frequencies of spontaneously apoptosing
lymphocytes. Our results of increased apoptosis have been recently
confirmed in two different animal models of MS by other authors.
Conclusion: Despite this impressive accumulation of evidences relating
the increase of apoptosis with the autoimmune activity of the disease
our results were considered to be very conflicting with the prevailing
paradigm of decreased T lymphocyte apoptosis as cause of increased
survival of autoreactive cells in MS patients. A new paradigm in which
excessive lymphocytes apoptosis can lead to homeostatic growth of
autoreactive cells has emerged.
129
MOLECULAR ORDERING OF THE FUNCTION OF
CASPASE 3 AND 6 BY SIMULTANEOUS MEASUREMENT
OF THE ACTIVITIES OF TWO CASPASES IN LIVING
CELLS WITH A NOVEL DUAL FRET INDICATOR PROBE
Xiaoli Wu1, James Simone2, Liusheng He3
1
National Institutes of Health (NIH), Bethesda, Maryland; 2Flow
Cytometry, NIAMS, NIH, Bethesda, Maryland; 3National Institutes
of Health (NIH), Autoimmunity Branch, National Institute of
Arthritis and Musculoskeletal and Skin Diseases (NIAMS),
Bethesda, Maryland
Downstream caspases-3 (DEVDase) and –6 (VEIDase) play complex
role in regulating apoptosis. Ordering the activity of these caspases has
been difficult. To explore the inter-relationship of caspase-3 and –6 we
developed a unique molecular probe to measure the activity of caspase3 and –6 in living cells simultaneously. This probe consists of a CFPYFP-mRFP fusion protein containing a caspase-3-cleavage motif,
DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID,
between YFP and mRFP. DEVDase and VEIDase activities could be
assessed simultaneously by monitoring diminished fluorescence
resonance energy transfer (FRET) mediated by cleavage of either or
both of these protease cleavage sites. DEVDase and VEIDase activities
were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and
enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD
and VEID cleavage specificities were validated by using caspase-3deficient MCF7-Fas cells and a caspase 6-specific inhibitor. Kinetic
analysis with the FRET probe revealed that caspase-3 activation
consistently preceded caspase-6 by approximately 30 minutes following
induction of apoptosis. Importantly, although caspase-6 and caspase-3
could be activated independently of each other when the two were
activated coordinately, caspase-6 was uniformly downstream of caspase3. Simultaneous detection of two caspase activities using this probe has
clearly provided information of the ordering of caspase-3 and –6 in the
apoptotic pathway.
Background: Several works on autoreactive T lymphocytes from
multiple sclerosis (MS) patients demonstrated increased expression of
anti apoptotic genes and in vitro increased survival of these cells when
were compared with MBP reactive PB T cells from healthy controls.
125
130
SIMULTANEOUS MEASUREMENT OF APOPTOSIS AND
NUCLEAR TRANSLOCATION EVENTS USING THE
Thaddeus George1, Brian Hall1, David Basiji1, Cathleen
Zimmerman1, Keith Frost1, William Ortyn1, David J Perry1,
Richard Esposito1, Richard Bauer1, Philip Morrissey1
1
Apoptosis is an organized, tightly regulated process by which a cell
orchestrates its own destruction. Inappropriately low rates can lead to
cancer or autoimmunity while inappropriate high rates can result in
neurodegenerative disorders or immunodeficiency. Factors that induce
apoptosis initiate a cascade of signals that eventually result in the
activation of intracellular effector proteases, or caspases, that cleave
cellular targets, resulting in the hallmark appearance of apoptosis,
including cellular blebbing, nuclear condensation and fragmentation.
In contrast, factors that inhibit apoptosis activate nuclear translocation
of the transcription factor, NF-kB. Once in the nucleus, NF-kB regulates
the expression of genes that promote cell survival and proliferation,
immune system function and inflammation. Thus, agents that stimulate
caspase activation but inhibit nuclear translocation of NF-kB have great
potential for the treatment of autoimmune disorders and cancer. Nuclear
NF-kB translocation is assessed by microscope-based systems since signal
localization is imperative. Measurement of NF-kB localization has
historically been done on adherent cells with large cytoplasmic to nuclear
area ratios. However, most cells of the immune system and many
hematological tumors exist ‘in suspension´ (e.g., lymphocytes,
monocytes, granulocytes) and adapting them to plates often proves
difficult. In this study we introduce a method for measuring nuclear
localization of NF-kB in the THP-1 suspension cell line, which have
very low cytoplasm to nuclear area ratios.
Here we present the
simultaneous measurement of NF-kB translocation and cell death by the
protein kinase inhibitor staurosporine. The data are acquired on the
ImageStream imaging flow cytometer which provides up to six channels
of imagery for each cell. The ability to acquire multi-spectral images of
large numbers of cells combined with morphology-based algorithms
allowed for the multi-parameter assessment of cell death and NF-kB
translocation in the same data file.
used to correct the autofluorescence in the PE and APC bands on a cellby-cell basis. Results & Conclusion Figure 1 shows two representative
dot plots of “negative” and “positive” HSIL specimens. The figure
demonstrates that flow cytometry is the analytical method of choice
because malignant cervical cells appear as rare events, typically between
0.05% and 0.5%. The threshold, set arbitrarily at this stage of method
development, identifies the malignant cells in which both biomarkers
are overexpressed. Using Pap tests as the reference, the sensitivity and
specificity of the flow cytometry method to classify cervical specimens
into “negative” and “positive” are 100% and 93%, respectively. Although
the results were based on a limited number of specimens, this clinical
test trial demonstrated the promise of using multi-parameter flow
cytometry for biomarker-based cervical cancer screening.
Figure 1. Dot plots of PE (P16 INK4a) vs. APC (Mcm5)
immunofluorescence intensities of the cells in a “negative” (left)
and a “positive” HSIL cervical specimen (right). Each plot
contains about 75,000 cervical cells.
132
HER2/NEU HERCEPTIN BIOMARKER DEVELOPMENT
FOR THERANOSTIC MANAGEMENT OF BREAST
CANCER PATIENTS
Ed Luther1, Alexey Glazyrin2, William Geddie3, James
Eliason2
1
Cancer Biomarkers
131
BIOMARKER-BASED CERVICAL CANCER SCREENING
USING FLOW CYTOMETRY - A NOVEL APPROACH
Jian Ling1, Urs Wiederkehr2, Spring Cabiness3, Kenneth R.
Shroyer4, J. Paul Robinson5
1
Southwest Research Institute, Medical Systems Organization, San
Antonio, Texas; 2Cytolution, San Jose, California; 3Southwest
Research Institute, San Antonio, Texas; 4University of Colorado
Health Sciences Center, Pathology, Medicine, Aurora, Colorado;
5
Background The Pap test, introduced 50 years ago, is still the only
cervical cancer screening method available. Due to non-standardized
manual sample preparation, staining, and visual microscopic reading,
the procedure is labor-intensive and subjective. To overcome these
limitations, a fully automated instrument is being developed. The novel
system integrates a completely standardized pre-analytical sample
preparation process and a quantitative cell-by-cell analysis of biomarkers
by flow cytometry. This study investigates the feasibility of using flow
cytometry to categorize clinical cervical specimens based on biomarker
expression levels. Methods p16 INK4a , a cyclin-dependent kinase
inhibitor, and Mcm5, a cell cycle regulating protein from the
Minichromosome maintenance protein family, are the biomarkers with
the highest potential to identify malignant cervical cells. These two
complementary biomarkers are capable of increasing specificity when
combined. Thirty-two residual liquid-based specimens from routine
cervical cancer screening were involved in the study. They were
categorized as 15 “negative” and 17 “positive” (2 ASC-US, 1 LSIL, and
14 HSIL) in Pap tests. Each specimen was stained with a cocktail of PE
conjugated p16 INK4a and APC conjugated Mcm5 antibodies. Five
parameters (FSC, SSC, FITC, PE, and APC) were measured by flow
cytometry. The cell autofluorescence measured in the FITC band was
126
CompuCyte Corporation, Cambridge, Massachusetts; 2Asterand
Corporation, Detroit, Michigan; 3Princess Margaret Hospital,
Toronto, Ontario, Canada
Approximately 25% of breast cancers over-express the HER2/Neu gene,
measured by Immunohistochemistry (IHC) staining or FISH probe
counts. A new antibody-based therapy (Herceptin™) is highly effective
in these cases. Current methods of HER2/Neu evaluation are neither
cost-effective nor highly accurate; therefore it is desirable to find more
practical and effective predictive biomarkers. Validation of laser
scanning cytometry technology for biomarkers in place. We developed
techniques for automated objective analysis of IHC-labeled tissue
microarrays utilizing laser scanning cytometry. Standardized CAP survey
arrays stained for HER2/neu were evaluated and compared with results
of traditional methods. Excellent correlation between the automated
results, the pathologist´s evaluation and FISH probe spot counts was
achieved. Development of novel Herceptin treatment biomarkers.
Herceptin is humanized hybrid antibody containing human Fc fragment
and mouse variable region. It would be reasonable to assume that the
actual therapeutic Ab Herceptin may be a better primary antibody in
theranostic IHC tests for Herceptin therapy patient selection. We
developed a novel method of staining breast tissue with Herceptin,
overcoming a major challenge of human Fc fragment IHC staining.
Serial breast tumor TMA sections were stained for HER2/neu and
Herceptin. Most HER2/Neu-positive core elements also showed
Herceptin expression. Conversely, 9% of tissue core elements were
labeled with polyclonal anti-HER2/Neu but not with Herceptin. The
discordance suggests that binding specificity of Herceptin differs from
that of the xeno-antibodies. In 3% of cases, tissue was reactive to Herceptin
but not to polyclonal anti-HER2/Neu. The latter result could not be
explained by existing knowledge of polyclonal and monoclonal
antibodies specificity. We double-stained IHC tissue sections and TMAs
with HER2/Neu and Herceptin developed by DAB and BCIP/NBT to
clarify this phenomenon. Some tissues exhibited mosaic staining patterns,
with various cells positive for both markers and neighboring cells positive
for only one marker. Demonstrated mosaic staining of tumor tissues
may identify additional candidate patients for use of anti-HER2/Neu
therapeutic antibodies different from Herceptin-target peptide areas. A
number of these Ab are currently in clinical testing. Summary: We
demonstrated that Herceptin can effectively replace xeno-antibodies
for IHC-based patient selection for breast cancer therapy. Our data
suggests that Herceptin binds to a different epitope than traditional
HER2/Neu Abs, perhaps resulting in a more relevant specificity.
Automated laser scanning cytometry analysis was proven to be an
invaluable tool in objective tissue characterization.
133
THE IMPACT OF TRASTUZUMAB, OMNITARG, AND
CETUXIMAB ON BREAST CANCER CELL
PROLIFERATION DEPENDS ON EGFR/HER2COEXPRESSION
Gero Brockhoff1, Elisabeth Schmidt-Bruecken1, Ferdinand
Hofstaedter1, Simone Diermeier1
1
University of Regensburg, Regensburg, Bavaria, Germany
Aims: ErbB-receptor targeting therapeutic antibodies are known to
impair growth of tumor cells. However, the cellular mechanisms
responsible for inhibition are incompletely understood. Methods: We
used Her2-overexpressing breast cancer cell lines to dynamically evaluate
the effect of EGFR-targeting antibody Cetuximab and Her2-targeting
Trastuzumab and Pertuzumab on cell proliferation in the presence and
absence of the growth factors EGF and HRG, showing different erbBreceptor binding specificities. Western Blotting was applied to investigate
receptor activation. The fraction of quiescent cells was quantified flow
cytometrically by BrdU/Hoechst-Quenching. The capacity of EGFR to
modulate the effect of Trastuzumab was demonstrated by anti-EGFRsiRNA. Results: Trastuzumab and Pertuzumab, targeted to Her2, turned
out to inhibit cell proliferation of BT474 and SK-BR-3 more efficiently
than Cetuximab. HRG strongly stimulates cell proliferation in both cell
lines and significantly compensates the inhibitory effect of Trastuzumab
and Pertuzumab. Proliferation was differentially modulated both in
G1- and G0-phase. EGFR content was efficiently downregulated by
SK-BR-3 cell transfection with anti-EGFR-siRNA and causes a BT474
like cellular behavior. Conclusion: Cell susceptibility to erbB-specific
antibody treatment in vitro is substantially determined by EGFR-HER2
coexpression and is additionally modulated by growth factors.
Consequently, erbB-receptor based strategies, dedicated to inhibit tumor
cell proliferation, have to account for erbB-receptor coexpression
134
ANTIGEN MAPPING IN MYELODYSPLASIA:
ABNORMALITIES OF CD34 AND CD117 EXPRESSION
ARE COMMON
Samuel J. Pirruccello1, Ken HE Young2, Patricia Aoun1
1
University of Nebraska Medical Center, Pathology and
Microbiology, Omaha, Nebraska; 2University of WisconsinMadison School of Medicine, Pathology and Laboratory Medicine,
Madison, Wisconsin
Introduction of five or more parameter flow cytometers into the clinical
laboratory has greatly enhanced the resolution and sensitivity of flow
cytometry, but has complicated data analysis when using conventional
population gating techniques. This is especially true for
myeloproliferative and myelodysplastic processes where multiple cell
lineages and stages of cell differentiation are present. We developed an
alternative method of data analysis, termed antigen mapping, that utilizes
antigen defined color gating to create more complex antigen distribution
patterns on the CD45 by side light scatter histogram (primary antigen
map). We utilized this analytical approach to elucidate recurring
myeloblast phenotypic abnormalities in a series of 28 myelodysplastic
bone marrow specimens including 8 cases of refractory anemia with
ringed sideroblasts (RARS), 8 cases of refractory anemia with multilineage dysplasia (RCMLD) and 12 cases of refractory anemia with
excess blasts (RAEB). The goals of the study were: (1) to utilize the
antigen mapping approach to define and classify common myeloblast
immunophenotypic abnormalities useful for phenotypic diagnosis of
MDS; and (2) to correlate the number and types of phenotypic
abnormalities observed with MDS grade, International Prognosis Scoring
System (IPSS) scores and IPSS risk classification. A total of 13 parameters
were measured on the myeloblast population including; side light scatter,
CD13, CD33, CD34, CD45 and CD117 density, increased expression of
myeloblast CD4dim and CD11c and aberrant expression of CD7dim
and CD56. A myeloblast phenotypic score was determined by summing
the total number of phenotypic abnormalities. The presence of decreased
myeloblast CD45 density, increased CD13 and CD34 density and
increased expression of CD11c and CD4dim were MDS grade related.
We observed a direct relationship between the number of myeloblast
phenotypic abnormalities (phenotypic score) and MDS grade. The
myeloblast phenotypic scores were also highly correlated with
International Prognostic Scoring System (IPSS) scores and IPSS risk
categories. Abnormal CD34 and CD117 bi-variate expression patterns
were highly informative being present in 50% of RARS, 68% of RCMLD
and 100% of RAEB cases. We identified several recurring abnormal
expression patterns of CD34 and CD117 that we have subsequently
observed in both myelodysplastic and myeloproliferative disorders. We
found the antigen mapping technique to be an informative and efficient
method of data presentation for detection of MDS associated
abnormalities of antigen distribution and density. In a high percentage
of the RCMLD and in all of the RAEB cases, the presence of a
myelodysplastic process could be deduced by visualization of the
primary antigen maps alone.
135
MONITORING MOLECULAR TARGETED
THERAPEUTICS IN PERIPHERAL BLOOD SAMPLES
FROM ACUTE LEUKEMIA PATIENTS BY FLOW
CYTOMETRY
Sue Chow1, Frances Tong2, David Hedley3
1
Princess Margaret Hospital/Ontario Cancer Institute, Applied
Molecular Oncology, Toronto, Ontario, Canada; 2Princess Margaret
Hospital/Ontario Cancer Institute, Toronto, Ontario, Canada;
3
Signal transduction analysis by flow cytometry is emerging as a new
and potentially powerful clinical application. Our own interest has
been in the development of techniques that can be used to monitor
pharmacodynamic effects of novel anticancer agents in clinical trial.
This creates significant technical challenges, since the assays require
whole blood processing to maintain drug/target equilibrium, and ability
to identify subpopulations of cells that are often present at low numbers.
Following an extensive analysis of sample processing conditions, we
recently published a technique that measures activated intracellular
signaling elements while maintaining light scatter and surface
immunophenotype (Chow et al. Cytometry 2005;67A:4-17). Based on
this, we developed a protocol that measures signaling pathways
downstream from growth factor receptors in AML blasts, including
ERK, PI3-kinase/Akt, and mTOR pathways. Using fresh whole blood
samples, we found a high degree of heterogeneity in constitutive signaling
activity in unstimulated AML blasts, and assessment of the clinical
significance of this is ongoing. An important aspect of this technique is
that it is sensitive to the effects of a wide range of pharmacological
inhibitors currently under clinical trial. Because standard phenotypic
features used to identify leukemic blasts are well preserved during sample
preparation, it is possible to measure signaling pathways in individual
cells present at very low frequencies, offering the potential to track
alterations in minimal residual disease. The protocol is sufficiently
rapid to allow same-day sample turnaround, and could therefore be
used for real time monitoring of pharmacodynamic effects in leukemia
patients.
136
ANALYSIS OF VARIATION IN RESULTS OF FLOW
CYTOMETRIC ZAP-70 EXPRESSION ON B AND T
LYMPHOCYTES IN A MULTICENTER STUDY
Jaco Kraan1
1
Erasmus MC - Daniel den Hoed Cancer Center, Internal Oncology,
Rotterdam, Netherlands
On behalf of the Flow Cytometry Working Party of SIHON (Foundation
for Immunophenotyping of Hematological Malignancies in The
Netherlands) Initially, ZAP-70 expression on B CLL cells has been
reported as percentage of positive cells with a cut-off based on T
lymphocytes (method 1), whilst others used a cut-off based on an isotype
control or on the ratio of mean fluorescence intensity (MFI) between
stained cells and a negative control. We performed a multicenter study
(n=12) comparing these 3 strategies to quantify ZAP-70 expression
using a 4-color, 5-antibody assay (ZAP-70, CD3 +5, CD19, CD45) and
two ZAP-70 antibodies conjugated with either Alexa 488 or PE. Our
127
results show that differences between centers for setting correction of
spectral overlap has an important effect on all methods which use the
ratio of ZAP-70 expression levels by B and T cells. This effect of
instrument setup is caused by differences between centers with respect
to the amount of spectral overlap into the ZAP-70 channel per instrument
and per lymphocyte subset stained with another fluorochrome. This
situation becomes particularly critical when B or T cells are conjugated
with a fluorochrome that has a relative large spill over into a channel
used to detect ZAP-70 (e.g. CD3 FITC vs ZAP-70 PE). Based on the
results shown in the table, we conclude that ZAP-70 expression reported
as the MFI ratio between B cells stained with ZAP-70 conjugated with
Alexa 488, and unstained B cells, is associated with the smallest interlab
variation, and therefore appears to be the most robust method for
multicenter studies
Interlab CV for 3 reporting methods for ZAP-70 expression on B
lymphocytes
ZAP-70
ZAP-70
ZAP-70
ZAP-70
PE
PE
Alexa 488
Alexa 488
ZAP-70
Reporting
method
Sample 1 Sample 2
Sample 1
Sample 2
% ZAP-70
on B cells
76%
51%
68%
58%
MFI ratio
B/T cells
76%
49%
38%
38%
MFI ratio
B cells
47%
56%
13%
15%
Environmental, Marine and Microbiology
137
SHORT-TERM VARIATION OF THE PHYTOPLANKTON
ASSEMBLAGE IN THE BAY OF MARSEILLE (FRANCE)
MONITORED BY IN SITU FLOW CYTOMETRY
Melilotus Thyssen1, Michel Denis2
1
Centre d’Océanologie de Marseille, Laboratoire de Microbiologie,
Géochimie et Ecologie Marine, UMR6117, Marseille Cedex 9,
France; 2Centre d’Océanologie, Marseille Cedex 9, France
High frequency single cell analysis is of great importance in monitoring
short-term ecosystem changes due to human activity or natural events.
Phytoplankton communities may be crucial in understanding coastal
systems because they encompass a wide spectrum of biological
information. Due to their large biodiversity and fast growth rate, they
exhibit quick responses to environmental changes in terms of
composition, succession patterns, abundances, and activity. These shortterm variations of phytoplankton community are poorly documented
because of field sampling limitations and the lengthy observation time
required by optical microscopy. With such conditions, the use of
phytoplankton in assessing the quality of the coastal waters remains
very limited. To avoid these technical difficulties and achieve
observations at a short-time scale, we moored a submersible flow
cytometer (CytoSub, CytoBuoy b.v., Bodegraven, The Netherlands)
and investigated the short-term variability of phytoplankton in a sailing
harbor of Marseille (Northwestern Mediterranean Sea). This instrument
can analyse phytoplankton in the size range 1-1000 µm with a time
interval as short as 10 min and a high flow rate, enabling detection of
single cells, chains and “rare” events. Phytoplankton was thus monitored
in situ at a fixed site, 1.5 m depth, over two months during summer
2005. Seawater was analysed every 30 min. The data treatment was
conducted on the basis of pulse-shape analysis, a procedure that enables
collection of much more information than usual softwares based on
peak-intensity or peak-area analysis. Up to 13 subpopulations were
thus identified. Daily sampling of nutrients (NO3, NO2, PO4) and
continuous information on salinity, temperature, fluorescence, wind
speed and global light intensity allowed distinguishing between the
impact of environmental factors and light/dark cycles on phytoplankton
communities.
128
138
PRELIMINARY INVESTIGATION OF
SYNECHOCOCCUS SP. AND PROCHLOROCOCCUS SP.
OF SOUTH WESTERN WESTERN AUSTRALIA
Harriet Paterson1, Kathryn Heel-Miller2
1
University of Western Australia, Animal Biology, Crawley, Western
Australia, Australia; 2University of Western Australia, Biomedical
Imaging & Analysis Facility, Faculty of Medicine and Dentistry,
Crawley, Western Australia, Australia
The west coast of Australia represents a unique environment in which to
study marine organisms as the major current, the Leeuwin Current,
flows pole-wards rather than equator-wards like all other eastern
boundary currents. This current is stronger in winter and during summer
the coastal Capes Currents develops. These currents influence the physical
and chemical properties of the water column and thus the distribution
of picoplankton in general. The data presented here represents the first
quantification of picoplankton on the coast of Western Australia using
flow cytometry and is composed of two sections. Firstly, as part of the
Strategic Research Fund for the Marine Environment, three sites were
sampled to determine microzooplankton grazing, on an inshore/offshore
transect, evaluating their bactivory on Synechococcus sp. and
Prochlorococcus sp. In addition samples were collected from the long
term CSIRO monitoring station west of Rottnest Island to examine the
vertical distribution of Synechococcus sp. and Prochlorococcus sp.
The abundance of Synechococcus sp. and Prochlorococcus sp. were
within the lower ranges reported in the literature, whilst the transect
stations had an order of magnitude more cells than the RNI station.
Carbon was also within the lower ranges reported in the literature.
Synechococcus sp. and Prochlorococcus sp. were both grazed at >100%
across the transect. At the inshore station, Synechococcus sp. experienced
exceptional grazing pressure. Grazing pressure was responsible for
preventing accumulation of these species in this region, both onshore
and offshore. Synechococcus sp. was more abundant than
Prochlorococcus sp. at Rottnest Island (RNI) station. The vertical
distribution of both species showed a maxima at 30 m depth below the
thermocline and in approximately 4-5% surface irradiance These
preliminary findings suggest that Synechococcus sp. and
Prochlorococcus sp. are found in similar quantities to other oligotrophic
regions of the world and contribute to the microbial foodweb.
139
CHARACTERIZATION OF ACANTHAMOEBA–
MICROSPHERE ASSOCIATION BY MULTI-PARAMETER
FLOW CYTOMETRY AND CONFOCAL MICROSCOPY
Christopher J. Hewitt1, Steve Smith2
1
University of Birmingham, Chemical Engineering, Engineering,
Edgbaston, United Kingdom; 2University of Aston, Life and Health
Sciences, Birmingham, United Kingdom
Acanthamoebae are members of a group termed small free-living
amoebae (FLA). Once known as soil amoebae owing to their presence
in moist soils, they are also found and are ubiquitous in a wide range of
aquatic habitats, both natural and man-made. Commonly members of
biofilm communities, actively predating bacteria, they also harbour
bacterial pathogens. Amoebae provide such pathogens with a suitable
environment for proliferation and dissemination, and may also support
the selection of facets, such as inhibition of lysosomal fusion, which in
turn allow pathogenic bacteria to avoid destruction by human
macrophages. Furthermore, acanthamoebae may also be considered
pathogens in their own right, mediating painful and debilitating diseases,
such as acanthamoebal keratitis, and even life-threatening infections.
Acanthamoebae in common with other protozoa readily phagocytose
particulate material, which in turn may lead to the spread of infectious
disease. Evaluation and quantification of plain and carboxylate FITC–
microsphere association with acanthamoebal trophzoites was undertaken
using a combination of multi-parameter flow cytometry and confocal
microscopy. Trophozoites from strains and species of Acanthamoeba
were exposed to plain and carboxylate FITC–microspheres. Microsphere
size and aspects such as trophozoite starvation, maturity and exposure
to metabolic inhibitors were assessed. All species and strains of
Acanthamoeba readily associated with plain and carboxylate
microspheres. Starving trophozoites significantly increased binding and
potential ingestion of microspheres, while trophozoites of increasing
maturity lost such abilities. Trophozoites showed a significant preference
for 2.0-ìm-diameter microspheres compared with other sizes, which
appeared to occupy much of the cytoplasm. The physiological inhibitors
sodium azide, 2,4-dinitrophenol and cytochalasin B reduced microsphere
association with trophozoites; however, some microspheres still bound
and associated with trophozoites after inhibitor exposure, a manifestation
of both active and inactive agent involvement in microsphere
phagocytosis. While the origins of microsphere binding by acanthamoebal
trophozoites remains shrouded, the combination of multi-parameter
flow cytometry and confocal microscopy supported synergistic
quantification and qualification of trophozoite–microsphere association
and ingestion.
140
POPULATION DYNAMICS WITHIN A MICROBIAL
CONSORTIUM DURING GROWTH ON DIESEL FUEL IN
SALINE ENVIRONMENTS
Susann Müller1, Sabine Kleinsteuber1, Ingo Fetzer1, Hauke
Harms1
1
UFZ- Centre for Environmental Research, Environmental
Microbiology, Leipzig, Saxonia, Germany
The diversity and dynamics of a bacterial community extracted from an
exploited oilfield with high natural soil salinity near Comodoro Rivadavia
in Patagonia (Argentina) were investigated. Community shifts during
long-term incubation with diesel fuel at four salinities between 0 and
20% NaCl were monitored by single strand conformation polymorphism
(SSCP) community fingerprinting of the PCR-amplified V4-V5 region
of the 16S rRNA genes. Information obtained by this qualitative
approach was extended by a flow cytometric analysis (MoFlo,
DakoCytomation, USA) of the communities diverging at different
salinities. Conspicuous patterns of DNA contents indicating cell cycle
activity and narrow ranges of cell sizes were used to identify growing
subcommunities, to follow their dynamics and to separate them physically
by high resolution cell sorting for subsequent phylogenetic identification
by 16S rDNA sequencing. Reduced salinity favored the dominance of
Sphingomonas spp. whereas at elevated salinities Ralstonia spp. and a
number of halophilic genera including Halomonas, Dietzia and
Alcanivorax were identified. The combination of cytometric sorting
with molecular methods of community analysis allowed the detailed
monitoring of community adaptation and the identification of
metabolically active community members.
141
MONITORING OF ANTI-MICROBIAL PROPERTIES OF
SPICE EXTRACTS AGAINST FOOD SPOILAGE
BACTERIA USING FLOW CYTOMETRY
Mercedes Alonso-Gomez1, Martin Gerard Wilkinson1, Edel
Durack1
1
University of Limerick, Life Sciences, Castletroy, Co. Limerick,
Ireland
Spices are included in foods to impart flavour and potentially to inhibit
spoilage or pathogenic bacteria. However, little information is available
on the anti-microbial effects of particular spices, their spectrum of
activity and mechanism of action.
This study reports on the evaluation
of the anti-microbial effects of ethanol or water extracts derived from
commercial spice preparations against Bacillus subtilis , Escherichia
coli , Pseudomonas fluorescens , Listeria innocua . Extracts at
concentrations from 250 mg/L to 1.5 mg/L of onion powder, white
pepper, black pepper, nutmeg, chilli, ginger, garlic, and salt were evaluated
against 24 h cultures of bacteria during a 4 h incubation period at 30° C.
Controls consisted of untreated cells or cell suspensions with spice extract
solvents. Anti-microbial activity was monitored by; (i) mean change in
optical density (OD) at 600nm, (ii) viability staining by PI and Syto9
and analysis by flow cytometry (FCM), and (iii) recovery of treated
cultures on agar plates. OD of cultures exposed to spice extracts was
monitored continuously over 4 h and results were expressed as the
change in mean velocity (Ä OD/min). FCM analysis was carried out
after incubation to assess viability and cell damage. Damage was
determined by comparison with FCM profiles obtained after treatment
of cells with permeabilizing agent, CTAB, or bactericidal treatments
(70% (v/v) iso-propyl alcohol (IPA) or heating at 85°C). Samples of
cultures after incubation were spotted onto Tryptone-Soya agar (TSA)
to assess the recovery of cell viability. Change in mean velocity (d OD/
min) of cultures exposed to water soluble extracts indicated little antimicrobial activity except for chilli and garlic extracts. FCM profiles
from cells exposed to the water soluble extracts indicated little cell
damage even at highest spice concentrations but some degree of cell
permeabilization was observed after exposure to water soluble chilli
extracts. Growth on TSA occurred for all strains exposed to the water
soluble extracts indicating survival. In contrast, ethanol soluble extracts
exhibited varying degrees of anti-microbial activity. Differential activity
of individual spices was noted e.g white pepper against E. coli and
nutmeg against L.innocua. However, black pepper appeared to have
a strong anti-microbial effect against all test strains. FCM profiles
indicated dose–response effects in the degree of cell damage and
permeability. In agreement with OD and FCM, survival of strains did
not occur on TSA. FCM appears to be a useful method along with
conventional microbiological techniques with which to assess the
bactericidal effects of spices for food safety applications.
142
INDIVIDUALS BEHAVE DIFFERENTLY – MULTIPARAMETER FLOW CYTOMETRY FOR MONITORING
BACILLUS CEREUS BATCH FERMENTATION
PROCESSES
Christopher J. Hewitt1, Godfrey L William2, Nichola Helen
Foxall1, Andrew Want1, Colin Thomas1
1
University of Birmingham, Edgbaston, Chemical Engineering,
Chemical Engineering, Edgbaston, United Kingdom; 2Molecular
Probes / Invitrogen, Eugene, Oregon
Microbiology is important to both human health and industry, therefore
many methods have been developed to count micro-organisms in the
process environment. Accurate measurements relating to cell
proliferation and viability are essential if informed decisions about a
process are to be made, since process performance will depend largely
upon cell number and individual cell physiological state. The advantages
of using cytometric techniques over the more traditional microbiological
analyses are well documented and the development of multi-parameter
flow cytometric techniques in laboratories around the world has led to
the functional classification of the physiological state of single celled
micro-organisms. This classification is often based on either 1) the
presence or absence of an intact fully polarised cytoplasmic membrane
and the transport systems across it or 2) energy dependent/independent
intacellular enzyme activities. Using all of these techniques it is possible
to resolve an individual microbial cells physiological state, beyond
culturability (the latter usually based on the measurement of number of
c.f.u./ml) to include metabolic activity enabling assessment of population
heterogeneity and dynamics. In this work we describe the advantages/
disadvantages of three well-known flow cytometric techniques for
measuring bacterial cell physiological state, namely PI/DiBAC4, PI/
DiOC6(3) (both measure cytoplasmic membrane potential/permeability)
and the RedoxSensor ™ Green kit (Molecular Probes/Invitrogen) on a
batch culture of the sporeformer Bacillus cereus. This organism was
chosen because of its particular sensitivity to culture conditions. All
three methods were found to work well with comparable results
throughout but the RedoxSensor ™ Green kit (Molecular Probes/
Invitrogen) may have advantages when endopsores as well as vegetative
cells are present in a culture.
HCDM Workshop
143
INTRACELLULAR MOLECULES FOR THE STUDY OF
TISSUE BIOPSIES
Theresa Marafioti1, David Y. Mason2
1
Oxford University John Radcliffe Hospital, Nuffield Dept of
Clinical Laboratory Sciences, Oxford, Oxon, United Kingdom;
2
University of Oxford John Radcliffe Hospital, Haematology,
Oxford, England, United Kingdom
The development of monoclonal antibodies in the last 30 years led to
the definition of large numbers of important surface molecules on human
white cells, and these have proved of great use in research and for
clinical purposes. More recently, numerous white cell-associated
components have been defined by an alternative route, namely gene
cloning, and many of these are intracellular constituents. Antibodies to
such molecules are available on an ever-increasing scale, and they are
particularly suitable for use by the haematopathologist since they
commonly (unlike traditional monoclonal antibodies) react with
routinely processed tissue biopsy samples. Systematic studies in the
129
authors’ laboratory have shown that many intracellular constituents
(e.g. signalling-associated molecules) are comparable to traditional
surface markers in terms of immunocytochemical labelling intensity
and lineage/maturation-association. Examples will be given of how a
variety of such markers can be applied to the study of human lymphoid
cell populations and lymphoma. It is also relatively simple to label two
or three molecules simultaneously in routine biopsy material, greatly
increasing the scope of immunohistological studies in a clinical setting.
References: Mason et al. (2000) Double immunofluorescence labelling
of routinely processed paraffin sections. J Pathol 191:452-461 Marafioti
et al. (2003) Expression of B-lymphocyte-associated transcription factors
in human T-cell neoplasms. Amer J Pathol 162:861-871 Marafioti et al.
(2003) Phenotype and genotype of interfollicular large B cells, a
subpopulation of lymphocytes often with dendritic morphology. Blood
102:2868-2876 Gellrich et al. (2004) Immunofluorescent and FISH
analysis of skin biopsies. Am J Dermatopath 26:242-247 Marafioti et al.
(2004) Expression of intracellular signaling molecules in classical and
lymphocyte predominance Hodgkin´s disease. Blood 103:188-193
Pozzobon et al. (2004) Intracellular signalling molecules as
immunohistochemical markers of normal and neoplastic human
leucocytes in routine biopsy samples. Br J Haematol 124:519-533
Marafioti et al. (2005) Expression pattern of intracellular leukocyteassociated proteins in primary mediastinal B cell lymphoma. Leukemia
19:856-861 Marafioti et al. (2005) The NFATc1 transcription factor is
widely expressed in white cells and translocates from the cytoplasm to
the nucleus in a subset of human lymphomas. Br J Haematol 128:333342 Tedoldi et al. (2005) Transmembrane adaptor molecules: A new
category of lymphoid cell markers. Blood Sept 13 [Epub ahead of
print]
role of this complex family goes beyond the simple recycling of
nucleotides. The story behind the CD38 gene family (CD38 and CD157)
is representative of a more general trend involving several nucleotidemetabolizing ectoenzymes, such as CD39 and CD26. Indeed, it appears
that all these enzymes evolved from very well conserved ancestors,
usually acquiring a membrane anchorage (either transmembrane or
GPI) and developing parallel and apparently independent functions.
Relevant in an immunological context is the acquisition of functions
classically associated with receptor activity, such as signal transduction.
These include calcium mobilization and protein tyrosine
phosphorylation. Another common trait is localization in membrane
lipid microdomains and physical and functional association with partners
specialized in signal transduction. The association and the involvement
of selected ectoenzymes with diseases suggested that CD38 and other
nucleotide-metabolizing ectoenzymes may play a role in leukocyte
trafficking (e.g., CD157) and immuno-modulation (e.g., CD39). A direct
role in the pathogenesis of diseases is also suggested by the involvement
of CD38 in directing the prognosis of chronic lymphocytic leukemia
(CLL).
144
MEMBERS OF THE CD150 (SLAM) FAMILY AS HUMAN
CELL DIFFERENTIATION MARKERS
Chronic persistent inflammation generally occurs in a site-specific
manner. While it has been demonstrated that an endothelial cell “postcode” exists that regulates tissue specific trafficking, the potential role
that stromal cells such as fibroblasts might play in the retention of
leucocytes within inflamed tissue has not been explored. As there is
currently a paucity of specific markers cells for cells of the mesenchymal
lineage., we have used a classical antibody approach and raised
monoclonal antibodies to stromal fibroblasts in an attempt to
chararacterize different subsets of mesenchymal cells/fibroblasts. This
paper describes the characterisation of these new reagents and also
describes the identification and isolation of a novel subset of CD45 +
stromal cells cells in various inflammatory disease states such as
rheumatoid arthritis and cirrhosis. Our findings suggest that a stomal
area post code, defined by fibroblasts exisits and is modified during the
transition from resolving to chronic persistent inflammation.
Pablo Engel1
1
University of Barcelona, Immunology Unit. Department Cellular
Biology and Pathology, Medical School., Barcelona, Barcelona,
Spain
The CD150 (SLAM) family consists of nine leukocyte cell-surface
proteins involved in leukocyte activation that belong to the
immunoglobulin (Ig) superfamily. Six members of this family: CD84,
CD150 (SLAM), CD229 (Ly9), CD244 (2B4), CD319 (CS1), and NTBA associate with adapter proteins SAP and EAT-2. SAP is a short
intracellular molecule that is mutated in humans with X-linked
lymphoproliferative disease. Receptors of this family regulate cytokine
production and cytotoxicity of lymphocytes and natural killer cells. In
this study, we analyze the expression of cell-surface receptors of CD150
family on several hematopoietic cell types using flow cytometry. We
describe a broader distribution of than previously reported and show
that they are differentially expressed on hematopoietic cells. Moreover,
we show that cell-surface receptors of the CD150 family were
differentially expressed among hematopoietic progenitors. Our data
show that CD150 family members are useful markers to distinguish and
isolate distinct hematopoietic cell subpopulations. The heterogeneous
expression of these receptors indicates that these molecules may play
non-redundant functions in the regulation of both innate and adaptive
immune responses. The data reported here may help to design new
strategies in order to unravel the functional role of these molecules in
the regulation of immune responses and in immunopathological
conditions.
145
CELL SURFACE ENZYMES, LEUKOCYTE
TRAFFICKING AND IMMUNE RESPONSES
Fabio Malavasi1
1
University of Torino Medical School, Torino, Italy, Genetics,
Biology and Biochemistry, Medicine, Torino, Italy
The plasma membrane of human cells hosts a relatively large number
(~5%) of molecules acting as enzymes apparently operating in an antieconomical manner. Nucleotide-metabolizing ectoenzymes constitute a
family within this larger family and are represented by a set of molecules
involved in the catabolism and scavenging of extracellular nucleotides.
This process results in the synthesis of compounds that play a critical
role in cell homeostasis and metabolism, suggesting that the physiological
130
146
GENERATION AND CHARACTERISATION OF STROMAL
SPECIFIC ANTIBODIES AND THE IDENTIFICATION OF
A NOVEL SUBSET OF CD45+ EXPRESSING
MESENCHYMAL CELLS
Christopher Dominic Buckley1
1
University of Birmingham, Edgbaston, Division of Immunity and
infectin, Birmingham, England, United Kingdom
147
TIME AND SPACE RESOLVED ANALYSIS OF FUNCTION
OF LEUKOCYTE ANTIGENS BY ULTRA-SENSITIVE
SINGLE MOLECULE IMAGING
Hannes Stockinger1
1
Medical University of Vienna, Vienna, , Austria
The knowledge of the molecular mechanisms underlying formation of
the immunological synapse and T cell activation is key to understand
the adaptive immune response. A number of cell surface receptors and
signaling molecules involved in this process were identified and
characterized by the use of monoclonal antibodies. Little is known,
however, about the dynamic of subcellular organization, interactions
and functions of these molecules. The deficiency in this information is
because of the lack of appropriate technology. During the past years we
developed new methodologies for imaging single molecules, because
single molecule analysis is the only technique that can provide dynamic
data. The major aim was to perform the studies in living cells under
non-invasive conditions, which we solved by ultra-sensitive fluorescence
microscopy. Following the motion of individual molecules during
different phases of T-cell stimulation in the area of the immunological
synapse we can now show that components of lipid rafts variably
associate with different receptors upon activation. Furthermore, detection
of single molecules allows analysis of the stoichiometry of molecular
complexes and the determination of their life-time. We are also able to
employ live fluorescence energy transfer to visualize spatio-temporal
activation of key signaling molecules during T cell activation. For these
studies we have constructed biosensors by incorporating both cyan and
yellow fluorescent proteins into T cell signaling molecules. In summary
by high sensitivity, precise positional accuracy and temporal resolution,
investigation of single molecules provides new information about
organization and function of molecules in cells. Supported by the
GEN-AU program of the Austrian Federal Ministry of Education, Science
and Culture, the Competence Center Biomolecular Therapeutics and the
Austrian Science Fund.
148
HUMAN CELL DIFFERENTIATION MOLECULES:
HCDM, THE SUCCESSOR TO HLDA
150/P2
ESTABLISHMENT OF ORAL CANCER CELL LINES &
IDENTIFICATION OF THEIR MOLECULAR PHENOTYPE
Wan-Tao Chen1, Ronggen He2, Xiaojian Zhou2, Ping Zhang1
1
Shanghai Second Medical University Affiliated Ninth People’s
Hospital,Shanghai Key Lab of Stomatology, Department of Oral and
Maxillofacial Surgery, Shanghai, China; 2Shanghai Second Medical
University Affiliated Ninth People’s Hospital, Department of Oral
and Maxillofacial Surgery, Shanghai, China
Bernadette Swart1, Heddy Zola1
1
Child Health Research Institute, Adelaide, South Australia,
Australia
The HLDA Workshops have been responsible for the discovery and
characterization of many biomarkers of the immune system, and the
development of the widely-accepted CD nomenclature system.
Antibodies validated by HLDA have found important roles as research
and diagnostic reagents, and the CD molecules are increasingly used as
therapeutic targets. Thanks to progress in molecular technologies,
antibodies are no longer the primary tool for biomarker discovery, and
the HLDA Council decided in 2004 to change its focus to validation of
antibodies and their use in research, diagnosis and therapy. This change
was signaled by a change to the name HCDM (Zola et al., 2005). In the
year since the 8th HLDA Workshop HCDM studies have focused on two
major aspects: Regulatory T cells and the establishment of techniques
to identify the target antigens for the many “orphan” antibodies that
remain uncharacterized from the 8 HLDA Workshops. These studies
are still ongoing at the time of Abstract submission. We plan to present
multi-laboratory comparative data using several antibodies against the
Treg-associated transcription factor FoxP3, which will be a candidate
for a CD designation. Studies on the phenotype of regulatory T cells
will be presented in a companion study. With regard to identification of
the targets for “orphan antibodies”, we are optimizing procedures for
immunoprecipitation followed by mass spectrometric analysis of protein
sequence.
Poster Presentations
149/P1
MULTICOLOR FLOW CYTOMETRIC ERBB RECEPTOR
AND DNA QUANTIFICATION IN BREAST CANCER
CELLS
Arabel Vollmann1, Gero Brockhoff1, Ferdinand Hofstaedter1
1
Bavaria, Germany
Aims: Abnormal Her2/neu receptor expression, DNA aneuploidy and
elevated S-phase fraction define the malignant growth potential of breast
cancer cells. Evidence is mounting that Her2/neu is not a single player
in the transduction of growth-promoting signals, but acts in concert
with other members of the ErbB receptor family.To elevate their
diagnostic value, we established a multicolor flow cytometric (MFC)
approach in order to quantify the entire ErbB-receptor coexpression
profile in breast cancer cells combined with quantitative DNA analysis.
Methods: Based on cytokeratin staining with PerCP in order to identify
epithelial tumor cells, erbB-receptors are stained using FITC, PE, APC
and APC-Cy7 labeled monoclonal antibodies. DNA was
stoichiometrically stained with DAPI and measured on a LSR-I flow
cytometer (BD Biosciences). Receptor quantification was compared with
established diagnostic tools. Results: Breast cancer cells lines with known
receptor expression facilitated reliable flow cytometric receptor
quantification. Immunohistochemical staining correlates well with
fluorescence intensity derived from flow cytometric measurements.
Conclusion: Multicolor flow cytometry is a promising tool to reliably
quantify erbB-receptor coexpression profile in combination with DNA
analysis. Disaggregation of primary tumor tissue can be performed
without significant loss of antigens. This approach is dedicated to be
used complementary to cytogenetic diagnostic.
Objective: Oral cancers are the most common neoplasm in Asia. The 5
years survival rate for the patients with oral cancers has reached about
64.7% reported by our department. So it needed to increase the survival
rate and life quality. To explore the mechanism on carcinogenesis and
to investigate the biological character for oral cancers, based on the
establishment of cell lines and animal models as well as identification of
their molecular phenotype, can help to select effective methods for
diagnosis and treatment. Material and methods: Cancer tissue culture
method was used to establish cell lines. Cell clone selecting, in vitro and
in vivo, was carried out to built the cell lines possessing high metastatic
feature to the lung or brain. Through exposed in chemotherapeutic
drug to build a drug resistant variant cell line. cDNA array testing was
used to identify the differential expression genes related to drug resistance,
metastasis behavior and immortalized feature. Affymetrix gene
expression chips were used to identify differentially-expressed genes in
oral squamous cell carcinoma from oral mucosa. Results: By now, we
have established nine cell lines for oral cancers, including Tca8113,
Tca/cisplatin, Tb, Acc-2, Acc-3, Acc-M, HIOEC, Rca-B, Rca-T. About
29 genes were involved in metastasis for oral salivary adenoid cystic
carcinoma, 14 genes down-regulated and 15 genes up-regulated. And
there were 66 genes related to drug resistance for cisplatin. Over forty
genes were involved in Lymphonode metastasis for oral squamous cell
carcinoma,and the signal pathway of NF-kappa B plays a important role
in the progress of metastasis.39 unique genes were found to have
differentially expressed on head and neck squamous cell carcinoma
from 22 pairs of samples. Conclusion: The cell lines of oral cancers
established by our laboratory have provided solid basis for investigating
the mechanism on carcinogenesis and identifying the genomic feature.
Using these cell lines and their animal models, we have made great
progress in preventing and treating oral carcinoma.
Key Words: oral
cancer; cell line; animal model; gene expression profile Acknowledge:
The research was supported by the grants: National Natural Science
Foundation of China, No. 30330580, 30300388, 30171014, 39770802
& 30000193.
151/P3
SCREENING AND IDENTIFICATION THE CISPLATINRESISTANCE RELATED GENES IN HUMAN ORAL
SQUAMOUS CELL CARCINOMA CELL LINE
Ping Zhang1, Wan-Tao Chen2, Xiaojian Zhou3, Qin Xu2,
Mingbin Zhang5, Weiliu Qiu2
1
Hospital,Shanghai Key Lab of Stomatology, Shanghai, China;
2
Hospital,Shanghai Key Lab of Stomatology, Department of Oral and
Maxillofacial Surgery, Shanghai, 200011, China; 3Shanghai Second
Medical University Affiliated Ninth People’s Hospital, shanghai,
200011, China
Purpose:It is important to screen and identify those cisplatin-resistant
related genes and expression profiles in cancer cells.The cisplatin-resistant
related genes could help illustrating the underlined reason of drug resistant
and directing the chemotherapy in clinic. So the purpose of this study
was to identify differentially expressed genes associated with acquisition
of cisplatin resistance in human tongue squamous cell carcinoma cells.
Experimental Design: First we established a cell line which was resistant
to cisplatin. Global gene expression analysis was performed between
the cisplatin-resistant tongue squamous cell line and sensitive parent
cell line, using Affymetrix HU95Av2 microarray. To valuate the
differentially expressed genes identified from the microarray study,
several interesting genes, including CCND1, CCND3, GST-Pi and Pgp,
were chosen for validation of genechip results and for further
investigation in 43 clinical samples of squamous cell carcinoma of
tongue. Results: We identified sixty-six genes, among which 38 genes
were up whereas 28 down in abundance differentially expressed between
131
the cisplatin-resistant and sensitive cell lines. Among those wellestablished mechanisms for cisplatin resistance, some new candidate
genes such as RECQL in DNA repair and MAP2K6 in MAP pathway
were identified to be related to cisplatin resistance while others such as
Pgp and GST-Pi were found unrelated. Furthermore, the up-regulated
CCND1 and down-regulated CCND3 expressions were considered to be
significantly related to cisplatin resistance, derived from both microarray
analysis of the cell lines and immunohistochemistry stainings in the
resistant and sensitive clinical samples. Conclusions: Our research
presents comprehensive gene information associated with cisplatin
resistance in human tongue squamous carcinoma cells.
Acknowledgements. Supported by the ‘The National Natura science
Foundation of China, Grant No. 30330580,30171014,30300388.
152/P4
MULTIPLEXED FLUORESCENCE-IN-SITUHYBRIDIZATION OF ERBB RECEPTOR STATUS
Andrea Sassen1, Simone Diermeier1, Arabel Vollmann1,
Stephan Schwarz1, Gero Brockhoff1
1
Bavaria, Germany
Aims: The erbB or HER family of receptor tyrosine kinases (EGFR/
erbB1, erbB2, erbB3, erbB4) constitutes a complex network, coupling
various extracellular ligands to intracellular signal transduction pathways.
The resulting cellular responses lead to e.g. increasing cell proliferation
and decreasing apoptosis rate. A dysregulation of the erbB receptors as
a result of gene amplification or protein overexpression has been
associated with cancer development. There is mounting evidence that
the cooperation between every family members has to be taken into
account. The aim of this study is to establish a diagnostic tool to quantify
the gene status and the protein expression of all erbB receptors in primary
tumor tissues in a reliable and repeatable manner. Methods: Multiplex
(M-FISH) was used to analyze gene and centromer signals of all four
HER receptors of breast cancer cell lines and primary tissue samples,
combined with DNA staining. Additionally, receptor status was
investigated with fluorescent antibodies. New DNA probes have been
developed. Results: The method of M-FISH is established for detecting
gene loci and centromers of each member of the HER family with
newly-created FISH DNA probes, simultaneous with the verification of
protein expression, respectively. Conclusions: Here we show that
detecting gene amplification as well as protein expression can be utilized
for the efficient and reliable determination of combined receptor
expression. Our results provide a basis for the development of a
quantitative, standardized diagnostic tool in the near future.
153/P5
ANALYSIS OF THE CYTOKINE PROFILE TH1/TH2
AGAINST THE HUMAN PAPILLOMAVIRUS TYPE 16 E7
ONCOPROTEIN IN PATIENTS WITH INVASIVE
CERVICAL CANCER RECEIVING RADIOTHERAPY
Felix G. Delgado1, Alba L Combita1, Maria A Cespedes1,
Alexander Rodriguez2, Maria M Bravo1
1
Instituto Nacional de Cancerologia, Bogotá (Colombia), Grupo de
Investigación en Biología del Cáncer, Bogotá, Colombia; 2Instituto
Nacional de Cancerología, Bogotá (Colombia), Grupo Clínica de
Ginecología, Bogotá, Colombia
The development of precancerous lesions and cervical cancer is
associated with and ineffective host immune response against the HPV
16 oncoproteins. An alteration of the Th1/Th2 responses against proteins
from HPV has been observed in patients with cervical cancer.
Nevertheless, there are not studies that describe the cellular immune
response in patients who have been treated with radiotherapy. In this
work, the frequencies of HPV-16 E7-specific T helper lymphocytes
expressing IFNã or IL-4 were determined in six healthy women, 12
patients with invasive cervical cancer before treatment and in 4 patients
after radiotherapy. 1 x 106 PBMCs were stimulated during 12 hours
with autologous HPV16 E7-pulsed monocyte-derived dendritic cells (DCs)
in a T-cell:DCs ratio of 10:1, or directly with 10 µg/ml of HPV16 E7
synthetic peptides (E751-70, E765-84 and E779-98). The cells were
stained for CD4, CD69 and intracellular cytokines (IFNã and IL-4) and
analyzed by flow cytometry. E7-specific CD4 + IFNã+ T lymphocytes
were detected in 2 of 6 healthy women (mean frequency of
0.045±0.005%). These women were seropositive to VLP L1 of HPV 16
132
indicating a previous infection with this virus. Two of 12 women with
cervical cancer showed E7-specific CD4 + IFNã+ T lymphocytes (mean
frequency of 0.080±0.030%) and also CD4+ IL-4+ T lymphocytes (mean
frequency of 0.025±0.005%) while three of 12 presented only E7specific CD4+ INFã+ T lymphocytes (mean frequency of 0.033±0.003%).
When the peptides were used as antigen, a higher response was observed
against E765-84 or E779-98 peptides and this response was similar to
observed when the protein was used. The pos-treatment analyses showed
that 3 of 4 patients displayed an increase of the frequency of E7specific CD4+ IFNã+ T lymphocytes after the stimulation with the E76584 and E779-98 peptides. Previously to treatment, one of these patients
did not show specific immune response and two showed a lower
frequencies of E7-specific CD4+ IFNã+ T lymphocytes (mean frequencies
before: 0.035±0.005%; mean frequencies after: 0.375±0.253%). These
results support the concept that the progression of the lesion could be
associated to an alteration of specific Th1/Th2 immune response.
Additionally, after treatment some patients showed an increase of
frequencies of E7-specific CD4 + IFNã+ T lymphocytes, indicating that
the specific immune response probably can be modified by the effect of
radiation therapy and could be associated with a better response to
treatment. This is a preliminary study and at the moment a higher
amount of samples is being analyzed. This work was supported by
COLCIENCIAS, Colombia. Research Grant 21010413021.
154/P6
FLOW CYTOMETRY FOR IMMUNOPHENOTYPING
BREAST, OVARY AND COLON CANCER
Rafael Nunez1, John Magenau2, Sergio Zamorano2,
Antonella Lostumbo2, Divyesh Mehta2
1
University of Illinois at Chicago, Hematology Oncology Section,
College of Medicine, Chicago, Illinois; 2University of Illinois at
Chicago, Hematology Oncology Section, Chicago, Illinois
Methods of determining prognosis in breast, ovary, and colon cancer
includes detection of molecular markers such as the estrogen receptor
(ER), progesterone receptor (PR), and HER-2/neu. These markers are
routinely assessed via immunohistochemistry (IHC) and Fluorescent
In-situ hybridization (FISH). Flow cytometry (FC) has not yet been
established for routine detection of these markers in tumors. We
performed FC on cells from fourteen ovary, five breast, and four colon
cancer tumors. FC was used to detect the presence of ER, PR, HER-2/
neu, Epidermal Growth Factor Receptor (EGFR), E-cadherin, RAS,
ERK and TUJ1. Cells obtained directly from tumoral tissue were
simultaneous analysized by cell cycle stage (G0, G2M, and > G2M) and
presence of antigenic markers. In ovarian tumors, 7 of 14 samples
demonstrated >50% of cells with her-2/neu expression in aneuploid
fractions (>G2M), 9 of 14 in G2M phase, and only 2 of 14 in G0 phase.
With respect to ER, 7 of 14 ovarian tumors exhibited >19% of cells in
G2M and >G2M fractions, while only 1 of 14 samples had >19% at G0.
The PR receptor was expressed in >19 % of cells in 8 of 14 (>G2M), 7
of 14 (G2M), and 2 of 14 (G0). In breast tumors, 4 of 5 tumors
demonstrated >50% expression of HER-2/neu in G2M and >G2M cell
phases, and only 2 of 5 demonstrated >50% her2neu expression in G0.
Three of five tumors had >19% cellular expression of ER in the >G2M
and G2M phases, while only one had >19% in G0. With PR, 3 of 5
breast tumors had >19% cellular expression in G0, G2M, and >G2M
phases. In colon tumors, 2 of 4 had >50% cellular expression of her2neu
in >G2M fractions, 4 of 4 in G2M, and only 1 of 4 in G0. Interestingly,
cell fractions containing G2M and > G2M DNA had > 50% cellular
expression for RAS and ERK in 2 of 4 colon tumors. No colon tumors
expressed RAS and ERK significantly in G0 cells.
We found that ER,
PR, and HER-2/neu marker expression were consistent with established
expression patterns. Additionally, these data point to specific expression
patterns that are associated with the cell cycle stage. In particular, we
note high expression levels of Her-2/nue, ER, and PR in aneuploid
(>G2M) cell populations. These results suggest that patterns of differential
expression can be readily assessed in tumor tissues using flow cytometry,
and has the potential for broad application to the study of all solid
tumors.
155/P7
THE ROLE OF CD44 IN THE MALIGNANT PHENOTYPE
OF A TRASTUZUMAB RESISTANT BREAST CANCER
CELL LINE
Zsuzsanna Palyi-Krekk1, Márk Barok1, Markku Tammi2,
Jorma Isola3, Gyorgy Vereb4, PéTer Nagy5, János Szöllõsi1
1
Biology, Debrecen, Hungary; 2University of Kuopio, Department of
Anatomy, Kuopio, Finland; 3University of Tampere, Finland,
Institute of Medical Technology, Tampere, Finland; 4University of
Debrecen Medical and Health Science Center, Debrecen, Hungary;
5
Biology, Medical and Health Science Center, Debrecen, Hungary
The CD44 hialuronan receptor is a transmembrane glycoprotein playing
a critical role in the adhesion, migration, invasion and survival of cells.
Trastuzumab (Herceptin), a monoclonal antibody against the ErbB2
tyrosine kinase receptor, shows a therapeutic effect against a fraction of
ErbB2-amplified breast tumors. Clinical resistance to trastuzumab exists,
but the mechanisms are largely unknown. It has been show that the
activation of ErbB2 and CD44 is interdependent, but the exact pathway
of their interaction is not clear. We investigated the expression profile of
CD44 and ErbB molecules on trastuzumab resistant cells lines derived
from breast (JIMT-1) and gastric cancer (MKN7) and on their trastuzumab
sensitive counterparts (SKBR3 breast cancer and N87 gastric cancer
cells). ErbB2 overexpression was detected on each of these cell lines.
On the other hand, CD44 overexpression was observed only on the
trastuzumab resistant ones. SCID mice were injected with JIMT-1 tumor
cells followed by trastuzumab treatment with different delay times. The
longer the delay was, the more trastuzumab resistant the JIMT-1
xenografts became. After sacrificing the mice, tissue sections of the
xenografts were stained for ErbB2, trastuzumab and CD44. We observed
an anti-correlation between the cell surface density of CD44 and the
trastuzumab binding to JIMT-1 cells, a finding which may be explained
by a role of CD44 in the trastuzumab induced down-regulation of
ErbB2. In vitro experiments showed that both high (1000 kDa) and low
(2 kDa) molecular weight, exogenously applied hialuronan increased
the internalization rate of trastuzumab in the JIMT-1 cell line. Multidrug
resistance is a potent barrier to effective, long term therapy in cancer
patients and it is frequently attributed to enhanced expression of
multidrug transporters and some receptors. Expression of multidrug
transporter MDR1 (P-glycoprotein) on JIMT-1 was shown by flow
cytometric analysis. JIMT-1 cells were more resistant to doxorubicin
and pumped the drug more efficiently than SKBR3 cells. Hyaluronan
decreased the efflux rate of doxorubucin in JIMT-1 which was paralleled
by a decreased survival of the cells. Elucidation of the role of CD44
overexpression in trastuzumab resistant cell lines may help to understand
the causes of therapeutic failures in patients with this type of breast
cancer.
156/P8
COMPARATIVE EFFECTS OF RESVERATROL,
RESVERATROL ANALOGUES AND VINEATROL ON THE
CELL CYCLE OF COLON TUMORAL CELL LINES
Dominique Delmas1, Anna-Kristina Marel1, Gérard Lizard2,
Norbert Latruffe1
1
GDR-CNRS 2583, IFR92 / LBMC, Université de Bourgogne,
Faculté des Sciences Gabriel, Dijon, France; 2Inserm U498/IFR100
- LBMC, CHU/Hôpital du Bocage - Faculté des Sciences Gabriel,
Dijon, France
Despite major advances in surgery, radiation therapy, and chemotherapy,
cancer remains one of the most common causes of death. Colorectal
cancer is one of the most frequent in the world with an incidence
estimated to 945,000 cases per year. Each year, about 33,000 new cases
of colorectal cancer arise in France. The discovery of resveratrol as a
chemopreventive agent in colon and other carcinomas offered renewed
interest in grape products and dietary supplements based on resveratrol.
This chemopreventive activity has been related to the ability of the
compound to inhibit cell proliferation, to promote cell differentiation,
to suppress angiogenesis and/or to induce cell death, mainly apoptosis.
We demonstrated that this protective effect could be related to the ability
of resveratrol to arrest cell cycle progression or to induce the redistribution
of death receptors in membrane rafts contributing to the molecule ability
to trigger apoptosis in colon carcinoma cells (Delmas D et al, J Biol
Chem 2003, 278: 41482-41490; Delmas D et al, Oncogene 2004, 23:
8979-8986). In this study, we compared the cytotoxicity of resveratrol
to that of other grape-derived polyphenols and their effects on the
colon cancer cell distribution in the cell cycle. We observed by flow
cytometry that the cytotoxic effects of chemically synthezised resveratrol
is equivalent to vine-shots isolated resveratrol and to its acetylated
derivative. On the other hand, a dimer of resveratrol, epsilon-viniferin
and its penta-acetate have a little effect on tumoral cell proliferation.
The antiproliferative effects of these compounds were correlated with
their effects on the cell cycle. Indeed, we observed that the treatment
with resveratrol, or with its acetate form, accumulates colon cancer cells
in the S phase whereas the dimer of resveratrol did not affect the cell
cycle. The treatment with a grape-derived polyphenol-enriched
preparation, vineatrol (16% resveratrol, 25% epsilon-viniferin)
(Actichem, France) led to a S phase accumulation of tumoral cells,
attributed to resveratrol and not to epsilon-viniferin. The effect of these
various polyphenols was studied in several colon cancer cell lines (HT29,
HCT116, SW620, SW480) which presented different sensitivities.
Acknowledgements: We are grateful to Jean-Claude Izard (Actichem,
35 Boulevard du Danemark, BP380, 82003 Montauban, France) for
the vineatrol, vine-shots isolated resveratrol, epsilon-viniferin and to its
acetylated derivatives.
157/P9
DIFFERENCES IN CELL CYCLE REGULATION AFTER
PLATINUM DERIVATIVES TREATMENT IN SENSITIVE
AND CISPLATIN RESISTANT OVARIAN CANCER CELL
LINES
Viktor Horváth1, Karel Soucek1, Lenka SvihalkovaSindlerova1, Jirina Hofmanova1, Petr Sova2, Alois KozubíK1
1
The Academy of Sciences of the Czech Republic, Institute of
Biophysics, Laboratory of Cytokinetics, Department of Comparative
Animal Physiology and General Zoology, Faculty of Science,
Masaryk University, Brno, Czec, Brno, Czech Republic; 2PLIVALachema a.s., Brno, Czech Republic
Ovarian cancer represents one of the leading cause of cancer-related
deaths in women. Cisplatin as one of the most potent antitumour agents
which display high efficiency in the treatment of ovarian cancer, is only
active in a limited range of cancer types, and its effects are often decreased
due to intrinsic or acquired resistance. LA-12 (currently under 1st phase
of clinical evaluation) is a novel octahedral platinum(IV) complex
containing a bulky hydrophobic ligand - adamantylamine. We
investigated the effects of LA-12 on cytokinetic parameters of ovarian
cancer cell lines with different sensitivity to cisplatin - A2780 (ciplatin
sensitive), A2780cis (acquired cisplatin resistance) and SK-OV-3 (intrinsic
cisplatin resistance) and compared them with those of cisplatin. Our
previous studies showed that LA-12 was able to overcome both the
acquired and the intrinsic cisplatin resistance [1,2]. This work is aimed
to characterize in more detail several signal transduction pathways that
are activated in response to exposure to the platinum-based DNA
damage-inducing agents. Flow cytometric analysis of cell cycle
distribution and DNA synthesis using 5-bromodeoxyuridine
incorporation revealed that cisplatin in A2780 cells caused primarily Sphase arrest, as LA-12, but it was shifted to G2/M at later time points(24
h), when decreased cdk-2-associated kinase activity were detected.
Western blot analysis indicated a concentration-dependent accumulation
of p53 protein which is implicated in modulation of cell cycle as the
result of cellular response to DNA damage. Equitoxic LA-12 and cisplatin
concentrations increased p53 protein levels already after 6 h of treatment
followed by increasing of p21 levels at 12-24 h time points in studied
cell lines, both significantly higher after cisplatin treatment. However,
we expanded our investigations to examine the effects of LA-12 or
cisplatin in A2780 and A2780cis cells on the protein expression of
either p53-targeted genes that have been shown to be important in the
cellular response to DNA damage including Bax, Gadd45, Mdm2 or
cell cycle regulatory genes such as Cyclin A, Cyclin B1 or Cdk-2. In
addition, we used flow cytometric bivariate analysis of DNA content
and expression of Cyclins A and B; and BrdU pulse-chase study for
evaluation the effects of LA-12 or cisplatin on the kinetics of the cell
cycle progression in ovarian cancer cell lines studied. Supported by the
IGA grant No. 1QS500040507 of the Academy of Sciences of the
Czech Republic and by the Ministry of Industry and Trade of the Czech
Republic, Contract No. PZ-Z2/29, “New Medicines for Cancer Therapy”.
1) Horváth V., Blanárová O., et al.: Gynecologic Oncology (2006), in
press 2) Kozubík A., Horváth V., et al.: Biochemical Pharmacology 69,
373 (2005)
133
158/P10
WHOLE GENOME SCREEN OF PRIMARY MELANOMAS
BY ARRAY CGH
Margit Balazs1, Andrea Treszl2, Zsuzsa Rakosy2, Ágnes
BéGány3, Roza Adany2
1
University of Debrecen, Faculty of Public Health, Department of
Preventive Medicine, Division of Biomarker Analysis, Debrecen,
Hungary; 2Debrecen, Hungary; 3University of Debrecen, Medical
and Health Science Center, Department of Dermatology, Debrecen,
Hungary
Array-based comparative genomic hybridization (aCGH) technique
provide new possibilities to detect and precisely map chromosomal
copy number changes of tumor samples on a high resolution scale,
without the need of in vitro cell culturing. The purpose of this study was
to evaluate and map at high-resolution the genetic targets that undergo
copy number alterations during melanoma progression. Our study
represents the first genome-wide copy number analysis of human
malignant melanoma by aCGH. The microarray platform we used
contained 2464 BAC clones (HumArray3.1) spotted in triplicate, with
an average spacing between clones of 1.4 Mb. High level amplifications
(log2Ratio > 1) were detected on 92 genomic clones. Log2Ratio > 2
were rare in the melanoma genome, detected only in 4 tumors, including
the 4p12-4p13, 1p11-12, 1p31, 1p36.3, 15q26.2, 15q25-q26, 15q26.2,
15qtel, 7p21.1, 11q13.4 sequences. Clear homozygous deletions of
single clones were seen on 9p21.3 and 10q26.1. There was a good
correlation between melanoma stages and the number of deleted clones
(stage II-III: n= 209, stage IV: n=293 and stage V: n=406). The tendency
for chromosome sequence gains were similar but the difference between
stage II-III and IV was less striking (n=209 and n=230, respectively),
however for stage V the number of gained clones was significantly
higher (n=406). The most frequent deletions were seen on 10q26.1
(67%), 2q22 (62%), 11q22 (57%), 9p21 and 10q25.3 (52%). The
most frequent gains were seen on 6p21.3 (64%), 7q22.1-q22.2 (64%),
12q13.2 (60%), and more than 50 % of tumors exhibited gains on
7q11.23, 11p12.2, 11q1312p13.3, 16q24, 17q24, 17q25, 17q25.3,
19q13, 20q11.2-q12, 20q13 and 22q13.1. Many of the frequent changes
seen by chromosomal CGH could be detected, however new, more
precisely mapped alterations were also discovered. This study expanded
the number of chromosomal alterations involved in melanoma
progression and highlighted new amplifications in the melanoma
genome.(Supported by OTKA 048750, Mecenatura 0017)
159/P11
TUBULIN TYROSINE LIGASE EXPRESSION
CORRESPONDS TO CHANGES IN THE TYROSINATION/
DETYROSINATION STATUS OF -TUBULIN IN PROSTATE
CANCER CELLS
Karel Soucek1, Anh D. Phung2, J. Chloe Bulinski3, Richart
W. Harper4, Michael T. McManus5, Jason P. Eserich2
1
Institute of Biophysics Academy of Science of Czech
RepublicInstitute of Biophysics Academy of Science of Czech
Republic, Brno, CZ, Czech Republic; 2University of California,
Davis, Internal Medicine, Davis, California; 3Columbia University,
Department of Physiology and Membrane Biology and Cancer
Center, School of Medicine, New York, New York; 4University of
California, Davis, Internal Medicine, Sacramento, California;
5
University of California, San Francisco, Department of
Microbiology and Immunology, UCSF Diabetes Center, San
Francisco, California
Prostate cancer is the second cause of cancer death among men in the
Western world. Defining the biomolecular changes associated with
prostate cancer is an important endeavor, and potentially will lead to
novel strategies for early diagnosis and treatment of prostate cancer.
Microtubules play important roles in many aspects of cell function, and
are targets for therapy in most forms of cancer, including prostate
cancer. Detyrosination/tyrosination of the C-terminus of á-tubulin is a
unique posttranslational modification where the C-terminal tyrosine is
cyclically removed by a carboxypeptidase and readded by a tubulin tyrosine
ligase (TTL). It has been shown that detyrosination/tyrosination cycle
of á-tubulin is associated with progression through the cell cycle and is
involved in cellular differentiation, the precise role of this
posttranslational modification in cancer is not known. Molecular
134
profiling of multiple á-tubulin posttranslational modifications revealed
several prostate cancer cell lines displaying decreased expression of
tubulin tyrosine ligase protein that was associated with markedly
increased elaboration of detyrosinated á-tubulin. Using lentiviralmediated transfection of a vector expressing TTL siRNA we were able
to stably suppress TTL protein expression and induce higher levels of
detyrosinated á-tubulin in non-cancerous prostate epithelial cells. These
results suggest that the dysregulated tubulin detyrosination/tyrosination
cycle is caused by decreased expression of TTL. Our results demonstrate
a useful tool for the study of function of this specific posttranslational
modification of á-tubulin (detyrosination) that will aid in the elucidation
of its role in cancer.
160/P12
CD31 (PECAM-1) AND CD38 ANTIGENS ARE COLOCALIZED ON THE CELL SURFACE OF HL-60 HUMAN
MYELOBLASTIC CELL LINE
Olivier Herault1, Nathalie Gallay1, Michel Degenne3, MarieThérèse Georget3, Jorge Domenech1, Christian Binet1
1
Université François-Rabelais et CHRU de Tours, Inserm ESPRI
EA3855, Faculte de Medecine, Tours, France; 2CHRU, Laboratoire
d’Hematologie, Tours, France
CD31 (PECAM-1) is a cell surface molecule notably expressed on
endothelial cells, platelets and neutrophils. It is also found on many
cancer cells, such as leukemic cells. CD31 has been identified as a
ligand for CD38, a transmembrane glycoprotein present notably on
immature myeloid cells. Studying CD31 and CD38 expression on marrow
blast cells of 86 patients suffering from de novo acute myeloblastic
leukemia (AML), we observed a correlation between the expression of
these two membrane antigens (p<0,0001) which could be explained by
a co-localization of these two molecules on the membrane of leukemic
cells. The aim of this study was to search a co-localization between
CD31 and CD38 on the HL-60 human myeloblastic cell line using
fluorescence resonance energy transfer (FRET) and co-capping
experiments. Concerning FRET analyzis, HL-60 cells were incubated at
4°C for 1 h with the anti-CD31 FITC-conjugated mAb (WM59) and the
anti-CD38 Cy3-conjugated mAb (OKT10, conjugateg using FluoroLink
mAb labelling kit® from Amersham Biosciences). After 2 washes,
immediate acquisition of 20000 events was performed on a
FACSCalibur® flow cytometer equipped with a 488 nm argon laser and
energy transfer was detected on FL3 channel. Concerning co-capping
experiments, cells were stained with the anti-CD38 purified mAb and
TRITC-labeled GaMIg. Samples were then moved to 37°C (40 min) to
induce co-capping before adding cold PBS plus 0.5 % BSA to block the
experiment. Counterstaining was performed at 4°C for 30 min with
biotin-conjugated anti-CD31 mAb and ExtrAvidin® FITC (Sigma). After
washing, cells were fixed (4% paraformaldehyde), settled on poly-Llysine-coated coverslips and analyzed with a DXM1200F® digital camera
(Nikon) fitted to a DMR® microscope (Leica), the images having been
collected using the Lucia® software (Nikon). In FRET experiments,
we observed a shift of the FL3-histogram of the cells stained with anti
CD31 FITC mAb, suggesting a co-localization between CD31 and CD38.
These results were reinforced by the co-capping experiments
demonstrating an association between CD31 and CD38, as shown by
the superposition of green (CD31) and red (CD38) staining inducing
yellow fluorescence at a pole of the cells. In conclusion, here we
demonstrate for the first time a co-localization between CD31 and its
ligand CD38 on myeloid leukemic cells, which could explain the
correlation between the expression of these two membrane antigens on
the surface of blast cells of de novo AML patients.
161/P13
SELECTION OF AGONISTIC ANTIBODIES TO A
RECEPTOR TYROSINE KINASE USING FLOW
CYTOMETRY-BASED ASSAYS FOR DETECTION OF
TOTAL TYROSINE PHOSPHORYLATION AND
RECEPTOR INTERNALIZATION
Paul Larsen1, Siew Schleyer2, John Kunich2, David
Bohmann1, Lynn Webster3, Eddie Bautista1, Jeff Hsu1, Judith
Abraham2, Masahisa Handa1
1
XOMA (US) LLC, Preclinical Research and Development,
Berkeley, California; 2Chiron Corporation, BioPharma Research,
Emeryville, California; 3XOMA (US) LLC, Cell and Analytical
Development, Berkeley, California
Receptor tyrosine kinases (RTKs) are well characterized with regard to
their involvement not only in normal processes such as cell proliferation
and differentiation, but also in pathological processes such as cancer.
From a therapeutic standpoint, stimulation of signaling through RTKs
has been pursued as a potential treatment for a variety of indications,
such as activation of trk RTKs for the treatment of neuromuscular
degeneration. In such cases, the development of agonist antibodies to
the RTKs represents a viable therapeutic option, and may be preferable
to ligand-based therapeutics due to the high affinity and long circulating
half life achievable with antibodies. To identify agonistic antibodies
targeted to one particular RTK of interest, we developed two flow
cytometry (FACS)-based assays to monitor downstream effects of
receptor activation: (1) total cellular phospho-tyrosine (pY), as a measure
of activation of signaling pathways; and (2) receptor internalization, to
measure down-regulation of activated receptor. The total cellular pY
assay employed a suspension-adapted, stably transfected CHO cell line
expressing high levels of the receptor. This assay was used to screen
hybridoma supernatants, purified hybridoma-derived antibodies, and
purified whole IgG reformatted phage display-derived antibodies.
Approximately 15% of the antibodies tested showed agonistic activity
in the pY assay. Immunoprecipitation followed by Western analysis
was then performed on lysates from antibody-treated cells (RTK overexpressing CHO and tumor cell lines), and the data confirmed that the
pY-inducing antibodies triggered phosphorylation of the target RTK.
Top candidates identified using the total cellular pY assay were further
characterized for cell surface RTK down-regulation and degradation.
Epitope competition studies were first conducted using FACS and
Biacore® to identify strong FACS-positive “detection” antibodies that
showed minimal competition with the top pY-inducing antibodies. These
antibodies were employed to develop a FACS-based assay monitoring
cell surface RTK levels from 2 to 72 hours after addition of pY-inducing
antibodies. A subset of the antibodies showed dramatic down-regulation
of cell surface receptor by 2 hours that was maintained through 72
hours. Western analysis confirmed that RTK down-regulation observed
in the FACS assay was associated with degradation of the RTK protein,
and also indicated that the drop in total cellular levels of the RTK
persisted for a greater period of time in response to the pY-inducing
antibodies than in response to the natural ligand.
162/P14
IDENTIFICATION OF NEUTRALIZING ANTIBODIES TO
A G PROTEIN-COUPLED RECEPTOR (GPCR) LIGAND
USING A FLOW CYTOMETRY-BASED INTRACELLULAR
CALCIUM FLUX ASSAY
Paul Larsen1, Amita Patel1, Elizabeth Pongo1, David
Bohmann1, Jody Brink1, Tamlyn Neben1, Marina Roell1,
Rhonda Hansen1, Steve Grimes2, Masahisa Handa1
1
XOMA (US) LLC, Preclinical Research and Development,
Berkeley, California; 2Aphton Corporation, Philadelphia,
Pennsylvania
G-Protein Coupled Receptors and their ligands have been implicated in
the development and progression of a variety of cancers, making them
potential targets for antibody-based therapeutics that neutralize their
function. One downstream signaling cascade that can be initiated upon
binding of a GPCR ligand to its cognate receptor is phospholipase C
gamma (PLC-ã)-mediated intracellular calcium flux. Therefore, in order
to identify neutralizing antibodies to a GPCR ligand that is putatively
involved in certain types of cancer, a flow cytometry-based assay was
developed to measure ligand-induced intracellular calcium flux in a cell
line stably transfected with the cognate receptor. Due to the rapid
response of the target cell line to ligand stimulation, the Cytek Time
Zero system was used for on-line injection of stimulus. Using this assay,
a panel of fully human phage display-derived ligand specific antibody
fragments was screened for ligand neutralization and compared to top
mouse hybridoma-derived monoclonal antibodies. Those showing greater
than 65% neutralization were reformatted to whole IgG molecules.
Reformatted antibodies were retested, and the top candidates further
analyzed to generate IC50 values. The most potent antibody identified
by the calcium flux assay also exhibited the highest affinity as measured
by Biacore® analysis, suggesting a correlation between potency and
affinity for this target. The top candidates were also tested for
neutralization of signaling through the MAP kinase pathway using an
ERK1/2 phosphorylation assay and showed similar potency profiles to
those seen in the calcium flux assay, further supporting the capacity of
these antibodies to neutralize ligand-mediated cell signaling. In summary,
we have utilized a flow cytometry-based intracellular calcium flux assay
to select a fully human, GPCR ligand-neutralizing antibody derived from
phage display technology that is competitive in function to a hybridomaderived lead mouse monoclonal antibody.
163/P15
HUMAN GLIOBLASTOMA CELL LINES ARE RESISTANT
TO RADIATION-INDUCED APOPTOSIS EXPRESS HIGH
LEVEL OF BCL-2 AND METALLOTHIONEIN AND DO
NOT DEPLETE GLUTATHION
Maria Giovanna Valente1, Claudio Panzarella1, Dario
Coletti2, Wolfgang Goehde3, Burkhard Greve3, Krzysztof
Jamroziak4, Piotr Smolewski4, Tadeusz Robak4, Laura
Teodori1
1
ENEA Casaccia, Biotec-med, Rome, Italy; 2University of Rome La
Sapienza, Histology and Medical Embryology, Rome, Italy;
3
University Hospital Münster, Münster, Germany; 4Medical
University of Lodz, Department of Haematology, Lodz, Poland,
Poland
BACKGROUND. Glioblastomas are frequent and aggressive adult
primary brain tumors, routinely treated by radiotherapy. However,
glioblastoma is one of the most radio-resistant tumors and the efficacy
of this therapeutic modality is often limited by a diminished susceptibility
of the irradiated cells to undergo apoptosis. Bcl-2 over-expression has
been often associated with tumour radio-resistance, but very little is
known regarding this mechanism in human glioblastoma cells. In
addition, the mechanism by which Bcl-2 oncogene expression inhibits
radiation-induced apoptosis and its relationship with oxidative stress
and with free radicals scavengers as metallothionein and glutathione
has been not reported so far. In the present study, we investigated whether
the content of Bcl2, metallothionein and glutathione are associated to
glioblastoma cell radio-resistance. MATERIAL AND METHODS:Two
glioblastoma cell line established by us from two stage III glioblastomas
were used in this study. Cells underwent 0.5, 1 and 2 Gy X rays exposure
followed by 4 and 12 hrs recovery time. Then, flow cytometry assays
were applied to compare several factors related to apoptosis and oxidative
stress between irradiated cells and cells not exposed to radiation. The
expression of Bcl-2 and metallothionein was assessed by staining with
mAbs Glutathione level was quantified by functional test with mBCl
and CMF-DA dyes. The level of oxidative stress was assessed by
functional DCFH-DA assay. Apoptosis was measured by Annexin V
method. All measurements were performed using PARTEC PAS Flow
Cytometer. Apoptosis frequency was also tested by fluorescent
microscopy. RESULTS: We found that both tested human glioblastoma
cell lines exhibited a resistance to administered doses of radiation as
confirmed by low frequency of apoptosis after 4 and 12 hours recovery
time points. Moreover, glioblastoma cells expressed high level of Bcl2 and metallothionein that were not affected by radiation exposure.
Also glutahione was not significantly depleted and we observed low
levels of oxidative stress associated with irradiation. CONCLUSION:
Our results suggest that radio-resistance of glioblastoma cells may be
related to interaction between Bcl-2 pathway and mechanisms involving
free radicals scavengers as metallothionein and glutathione.
135
164/P16
MULTIPARAMETER FLOW CYTOMETRIC ASSAYS FOR
THE STUDY OF APOPTOSIS
Carl Bortner1, Maria Sifre1, John Cidlowski1
1
National Institute of Environmental Health Sciences, Department of
Health and Human Services, National Institutes of Health,
Laboratory of Signal Transduction, Research Triangle Park, North
Carolina
Apoptosis is a physiological programmed cell death process defined by
a unique and classical set of morphological and biochemical
characteristics including the loss of cell volume or cell shrinkage,
chromatin condensation, internucleosomal DNA fragmentation, and
apoptotic body formation. Over the years, additional characteristics
have been observed that have been used to define this mode of cell
death including the externalization of phosphatidylserine, activation of
proteases known as caspases, cleavage of various apoptotic target
proteins, and specific changes in intracellular ions resulting in the loss
of cell volume. Flow cytometry has been a principal tool in defining
whether a cell is undergoing apoptosis. However many of these flowcytometric assays examine only a single or specific characteristic of the
apoptotic process limiting our understanding of this very dynamic and
complex process. An in-depth understanding of the physiology of
apoptosis requires knowledge of how each of these individual apoptotic
characteristics relates to each other. Thus, we have developed several
multi-parameter techniques for flow cytometry by combining individual
apoptotic assays such as the examination of intracellular ions, AnnexinV binding, and caspase activity to a single set of cells to distinguish the
impact one apoptotic characteristic has in relation to other events during
the programmed cell death process. This type of in-depth analysis of
apoptosis has begun to expand our appreciation of the orchestration of
events that comprise this physiologic cell death program.
165/P17
CELL DEATH IN LEUKOCYTES AND THE USE OF
1
1
1
Uriel Trahtemberg , Mizhir Atallah , Alon Krispin , Inna
Verbovetski1, Dror Mevorach1
Concentration and time dependent effects of calcium on apoptotic
monocytes.
Light scatter of apoptotic monocytes after 10 minutes incubation
with calcium in ice.
166/P18
REDUCTION OF SPONTANEOUS APOPTOSIS, BAK AND
BAX IN LARYNGEAL CARCINOMA
Gg Chen1, Ac Vlantis1, Ecw Chak1, Hc Liu1, Mcf Tong1, Ca
Van Hasselt1
1
Chinese University of Hong Kong, Surgery, Hong Kong, China
1
Hadassah University Hospital and The Hebrew University,
Medicine, Jerusalem, Israel
Annexin-V, which binds phosphatydil serine (PS) on the outer leaf of
the plasma membrane, has become one of the main apoptosis markers
used. This binding is known to be calcium-dependent. Calcium is a
central player in intracellular signaling, having a critical role in death
processes´ signaling. Thus we asked whether the use of calcium while
measuring apoptosis affects the apoptotic process itself. The literature
shows that Annexin-V apoptosis tests use calcium at 1.8 to 5 mM, most
commonly 2.5 mM, some articles not even mentioning the concentration
being used. In this work we tested the effect of different calcium
concentrations and incubation times on viable and apoptotic human
monocytes, neutrophils and dendritic cells. Titration of Annexin-V
showed that calcium concentrations as low as 1 mM produce results
similar to those of concentrations as high as 5 mM. In order to isolate
the requirement of calcium for Annexin-V binding from its pro-apoptotic
effect we used cells lightly fixed with paraformaldehyde and obtained
similar results. Most importantly, while kept in ice, as calcium
concentration and incubation time increase the percentage of apoptotic
and necrotic cells grows accordingly (fig. 1). After only 10 minutes in
media containing 1.75 or 2.5 mM calcium apoptotic cells show marked
changes in their light scatter characteristics (fig. 2). The effects we
describe show some variability depending on the cell type and its viability
state but they were seen in all the settings we tested for. We propose that
the use of Annexin-V binding as a marker of apoptosis of primary
leukocytes should be performed using lower concentrations of calcium,
between 1-1.5 mM, and that the exposure to calcium be restricted to 10
minutes. We present a new method for such testing which consists of
keeping the cells in a medium with low or no calcium and adding it only
10 minutes before acquisition together with Annexin-V itself. This not
only minimizes exposure to potentially disruptive calcium but also allows
for the use of Annexin-V for concurrent measure of surface molecules
and apoptosis on live cells, opening new possibilities for the study of
the dynamics of apoptosis.
136
The growth of neoplasia is determined by the proliferation and loss of
cells. The aim of this study is to determine the frequency of apoptosis in
laryngeal carcinomas and to examine its relationship to the expression
of Bcl-2 family proteins, which play an important role in the growth
and biologic behavior of tumors. The materials studied are 39 cases of
laryngeal caner tissues. Apoptotic cells were determined by the TUNEL
method.
The expression of Bak, Bax and Bcl-2 was
immunohistochemically examined. The relationships between apoptosis
and the Bak, Bax and Bcl-2 proteins were studied. The expression of
both Bak and Bax was frequently detectable in tumor tissues but the
levels of both were much lower when compared with non-tumor tissues.
However, the expression of Bcl-2 was not different between tumor and
non-tumor tissues. The frequency of spontaneous apoptosis was lower
in tumor tissues than in non-tumor tissues but it was not significantly
related to any of these three protein expression. It was found that the
expression of Bak was decreased in the moderately differentiated tumors
compared with well differentiated ones. In contrast to Bak, the level of
Bcl-2 was increased in the moderately differentiated tumors compared
with well differentiated ones. The result is in line with the finding
decreased Bak in tumor tissues and indicates that the reduction in Bak
may associate with more malignant laryngeal tumors. The lack of
correlations between apoptosis and the expression of Bcl-2 family
proteins suggests that the spontaneous apoptosis in laryngeal carcinoma
is a complicated process and that factors other than Bak, Bax and Bcl-2
should also participate it.
167/P19
MEASUREMENT OF APOPTOSIS IN GROWING CELL
POPULATIONS
David Diaz1, Alfredo Prieto1, Hugo Barcenilla1, Jorge
Monserrat1, Luis Chara1, Julio Chevarria1, Miguel Ángel
Sánchez1, Eduardo Reyes1, Melchor ÁLvarez-Mon2
1
Spain; 2University Hospital “Príncipe de Asturias”, Immune system
Diseases and Oncology Service, Alcala de Henares, Madrid, Spain
The ability to accurately determine the number and population of cells
undergoing apoptosis will allow the evaluation of new therapies targeted
at inducing or inhibiting apoptosis providing an early marker of
therapeutic outcome. However, to use the apoptosis as a marker in
making clinical decisions more accurate methods of measurement of
apoptosis have to be developed. In current flow cytometry methods
(apoptotic index, AI), AI has been used to measure the proportion of
apoptotic cells in relation to the total number of detectable cells in the
test tube. In a given cell population phenotypically defined by the
expression of one characteristic lineage antigen (LAg) AI represent the
proportion of apoptotic cells displaying a specific LAg within a
population of cells that remain unfragmented and retain the expression
of the LAg. This approach has two limitations. First, apoptotic cells
frequently lose, partially or even completely, the cell surface expression
of the LAg used for the identification of the cell subset and these LAg-+
cells can no longer be identified as members of the original LAg
population. Second, apoptotic cells fragment into apoptotic bodies that
later disintegrate leading to an underestimation of the percentage of
apoptotic cells if the debris is excluded from the gates for cell analyses,
or, alternatively, to the overestimation of apoptosis if several apoptotic
bodies derived from a single cell are misinterpreted as individual
apoptotic cells. These limitations warrant the development of a new
method that provide an estimate of the number of cells that have
undergone apoptosis and its relation to the number of seeded cells: the
apoptotic rate (AR). This method takes into account the early apoptotic
cells which have suffered LAg loss and late apoptotic cells that
fragmented into apoptotic bodies. A limitation of the AR is that it can
only be properly applied in time frames in which the in vitro cell
proliferation does not significantly alter the number of cells in the
culture. This time frame depends on the rate of proliferation of the
studied cells. If the cells do not proliferate then apoptosis can be measured
accurately by AR at 24 h. or even 48 h. of culture. When cells proliferate
vigorously is necessary to perform the apoptosis assays after shorter
periods of culture (3-6 h) to avoid interference of proliferative processes
on the quantification of cell loss by apoptosis. Methods that enumerate
apoptotic cells can be also combined with CFSE tracking method alone
or combined with DRAQ5 labeling to provide much more accurate
information than those which only provide relative proportions of
apoptotic cells and can be applied even in conditions in which apoptosis
and growth occur simultaneously.
168/P20
ABNORMAL FAS/FASL AND CASPASE-3-MEDIATED
APOPTOTIC SIGNALING PATHWAYS OF T
LYMPHOCYTE SUBSETS IN PATIENTS WITH SYSTEMIC
LUPUS ERYTHEMATOSUS
Lanlan Wang1, Bei Cai1, Xue Chen1, Jie Chen1, Weihua
Feng1, Honggang Wei1
1
West China Hospital of Sichuan University, Department of
Laboratory Medicine, chengdu, Sichuan Province, China
Objectives. To explore the apoptotic status of lymphocytes of SLE
patients and the relationships between Fas mediated signaling pathway
of T lymphocyte subsets in patients with SLE. Methods. Flow cytometry
was used to determine the percentage of apoptotic cells (FITC-AnnexinV
positive, PI negative) and necrotic cells (FITC-AnnexinV positive, PI
positive). Expression rates of Fas AFasL and intracellular expression
rates of activated caspase-3 were evaluated by two-color flow cytometry
analysis in peripheral T lymphocyte subsets of SLE patients. Thirtynine patients with SLE fulfilling the revised 1982 ACR criteria for the
classification of SLE were recruited. Patients were classified as having
active or inactive disease on the basis of the SLEDAI. Health control
included thirteen female volunteers. Results. Compared with health
control group, the percentages of apoptotic lymphocytes and necrotic
lymphocytes enhanced in patients with inactive or active SLE (P<0.05).
The percentages of apoptotic cells and necrotic cells were higher in
active patients than in inactive patients. The percentages of CD4+T cells
expressing Fas (P<0.05, active or inactive patients vs. control) increased
in SLE patients. In SLE patients the percentages of CD8 + T cells
expressing Fas slightly increased but the difference was no statistical
significance (P>0.05, active or inactive patients vs. control). The
percentages of CD4 +T or CD8 +T cells expressing FasL were higher in
patients with active or inactive disease than those in health control
(P<0.05). But there were no obvious differences of expression rates of
Fas and FasL on T cell subset between two disease groups (P>0.05). The
expression rate of activated caspase-3 in T cell subset of active SLE
patients was notably higher than inactive SLE patients and health control.
The expression rate of caspase-3 in T cell subset of inactive SLE patients
was slightly higher than that in control group (P>0.05). In all patients
the percentages of CD4+T cells expressing Fas and intracellular activated
caspase-3 were higher than those of CD8+T cells. Conclusions. Apoptotic
speed of T lymphocyte subset in SLE patients accelerated, Fas -mediated
pathways were especially important for CD4+T cells undergoing apoptosis
in SLE patients with active disease. Increased Fas expression resulted in
a higher susceptibility to Fas-mediated apoptosis, which contributed to
the increased levels of intracellular activated caspase-3 and accelerated
apoptosis of T lymphocytes. Key words: Fas GFasL Gcaspase-3 GT
lymphocyte subset; systemic lupus erythematosus
169/P21
FLUOROCHROME-LABELED INHIBITORS OF
CASPASES (FLICA) STAIN CELLS WITH FRAGMENTED,
CONDENSED AND SWOLLEN NUCLEI DURING 7KETOCHOLESTEROL-INDUCED CELL DEATH
Anne Vejux1, Thomas Montange1, Edmond Kahn2, GéRard
Lizard1
1
Inserm U498/IFR100 - LBMC, CHU/Hôpital du Bocage - Faculté
des Sciences Gabriel, Dijon, France; 2Inserm U678 - CHU PitiéSalpêtrière, Paris Cedex 13, France
Oxysterols are cholesterol oxide derivatives which are found at enhanced
level in atherosclerotic plaques. Some of them, especially 7ketocholesterol (7KC), are supposed to play key roles in the development
of atherosclerosis. We recently reported (Cytometry 2005, 64A: 87100) that 7KC-induced cell death is a complex phenomenom preceded
by a rapid accumulation of lipids. As 7KC-induced cell death is associated
with important morphological nuclear changes (condensation,
fragmentation, and swelling of the nuclei) (J Biochem Mol Toxicol
2005, 19: 311-326), we clarified the relationships between nuclear
morphology and caspase activation. To this end, caspase activity was
measured on untreated and 7KC (40 µg/ml, 18-24h)-treated human
promonocytic U937 cells with FLICA both by flow cytometry (FAM
Poly Caspases Assay kit, Molecular Probes) and by conventionnal and
confocal fluorescence microscopy (Image-iT Live Red Poly Caspases
detection kit (Molecular Probes), nuclear staining with Hoechst 33342
(10 µg/ml)). Noteworthy, the percentage of FLICA positive cells
identified by flow cytometry was correlated with those of cells with
(fragmented + condensed + swollen) nuclei. By fluorescence microscopy,
caspase activity was not only detected in cells with condensed/fragmented
nuclei (typical of apoptosis) but also in some cells with swollen nuclei
(evocating oncosis). Thus, under treatment with 7KC, we clearly
established that the percentage of apoptotic cells is lower when it is only
based on the morphological aspect of the nuclei than when it is
determined with FLICA. Therefore, our data underline that the percentage
of apoptotic cells may vary depending on the method used to identify
apoptotic cells. They also favor the hypothesis that some connexions
might exist between 7KC-induced apoptosis and 7KC-induced oncosis
and/or that some caspases might also be activated in certain forms of
oncosis.
170/P22
MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN
PLANT CELLS DURING AUTOPHAGY
Josh J. Conolly1, Iona E. Weir1, Bruce C. Baguley2
1
BioDiscovery NZ Ltd, Auckland, New Zealand; 2The University of
Auckland, Auckland, New Zealand
The ability of plant cells to survive during times of nutrient deficiency
requires major changes in metabolism and the recycling of non-essential
137
cellular compounds and organelles. Considering a plants inability to
extradite itself from the environmental stress, it seems likely that the
process of autophagy would be fundamental for the overall plant’s
survival. Autophagy is the process in which these components are
targeted for use by the cell and has been well documented in yeast and
mammalian cells. The role of autophagy in plant cell starvation and
death is still not fully understood. We have used a combination of flow
cytometry, microscopy and molecular techniques to explore the cellular
changes which occur during autophagy and to compare this to apoptosis.
Nicotiana tabacum cv. Bright Yellow-2 (BY-2) cell morphology was
altered by conditions of starvation. Sucrose starved cells became smaller,
globular, lost many transvacuolar strands and their nuclei became
marginalized. Cells starved of nitrates became large, elongated, with
fewer transvacuolar strands and developed amyloplasts (starch granules)
around the nucleus. During both carbon and nitrogen withdrawal, total
DNA content in BY-2 cell nuclei appeared to increase, then decrease
when stained with propidium iodide (PI) and measured with flow
cytometry. The increase was accompanied by an observed swelling in
these nuclei upon introduction of PI. This apparent shift in DNA content
was not seen when nuclei were stained with Hoechst 33342 (H342).
Intracellular free-calcium levels in the starved BY-2 cells were assessed
using the ratiometric probe Indo-1. Both nitrogen and carbon
deprivation caused a slight increase in the baseline Ca2+ levels, when
compared to cells grown in complete medium. The starved cells also
displayed greater susceptibility to the effects of the calcium ionophore
ionomycin. The methylation-state of BY-2 cell DNA was analysed
using methylation-sensitive restriction fingerprinting, comparing cells
grown in complete media with those starved of either sucrose or nitrates.
Genome wide changes in DNA methylation were observed in the starved
cells, suggesting the possible role of methylation in altered PI binding
during the starved state.
171/P23
THE INFLUENCE ON CELL CYCLE CHECKPOINTS AND
APOPTOSIS INDUCED BY DIFFERENT DOSAGES OF XRAY
1
1
1
1
Daxing Xie , Yongdong Feng , Peng Zhang , Jianhong Wu ,
Deding Tao1, Jianping Gong1
1
Huazhong University of Science and Technology, Tongji Cancer
Research Institute, Tongji Medical College, Wuhan, Hubei, China
[Abstract] Objective There exists two DNA damage checkpoints in
normal cells, which are G1-phase checkpoint and G2-phase checkpoint.
At present, we have little knowledge upon their difference of radiationsensitivity and apoptosis. Using Cyclins/DNA flow cytometry and API
assay, our study was designed to analyze the influence on these
checkpoints and apoptosis induced by different dosages of X-ray, which
tried to enrich the theory of cell cycle checkpoints and instruct the clinic
radiotherapy. Method MOLT-4 Cells (wide-type p53) irradiated by
different dosages of X-ray(2, 5,10, 20 Gy) were gathered at 0, 3, 6, 9h
respectively, then each was divided into two groups. In one group,
expression of cyclin E, A, and B1 were detected by Cyclins/DNA flow
cytometry; in the other group, cell apoptosis was detected by API and
Annexin V/PI assay. Results When MOLT-4 cells were irradiated by
low dosage of X-ray (2 or 5 Gy), levels of cyclin E decreased in G1/S
phse, and G2 Cyclins (cyclin A, B1) accumulated distinctly in G2 phase,
which indicated that G2 checkpoint is activated. Simultaneously,
apoptotic rate of MOLT-4 cells increased slowly (reached 9.5% at 9th
hour), and cell cycle specific apoptosis happened in G2 phase. However,
when cells were irradiated by high dosage of X-ray (10 or 20Gy), levels
of cyclin E increased and accumulated distinctly in G1/S phase, and G2
Cyclins (Cyclin A, B1) decreased gradually, which indicated that G1
checkpoint is activated. Apoptosis increased quickly (reached 15.06%
at third hour), and cell cycle specific apoptosis happened in G1 phase.
Conclusion Cyclins/DNA flow cytometry and API assay can quickly
and distinctly detected the influence on different checkpoints and
apoptosis induced by X-ray, and our results suggested that G2 checkpoint
is more sensitive than G1 checkpoint to response DNA damage and
trigger cell cycle specific apoptosis.
172/P24
WRONG TIME AND WRONG PLACE OF CDK
ACTIVATION FOR G1 APOPTOSIS IN LEUKAEMIA CELL
LINE AND HUMAN PBL
Jianhong Wu1, Yongdong Feng1, Xiaolan Li1, Daxing Xie1,
Deding Tao1, Junbo Hu1, Jianping Gong1
1
Although apoptosis (programmed cell death) and cell proliferation have
entirely opposite end-points, substantial evidence now indicates that
these two processes are mutually coordinated. This coordination is
essential to preserve homeostasis and genomic integrity. Not surprisingly,
loss of the coordination of these processes predisposes to many
pathological conditions, e.g. cancer or AIDS. The mechanisms
underlying this regulation are yet to be completely understood. It is
well known that CDK1 is a major component of cell cycle progression
engine. But, more than ten years ago, it has been proved that p34cdc2
is necessary for apoptosis in lymphoma cells. Then, several researches
indicated that CDK1 unscheduled activity could induce cells go to
apoptotic death. However, it is still unclear why the cells will spend
different time to enter apoptotic cell death program after they are
triggered to die, why CDK1 drive cells go to apoptotic cell death, but
finish cell divide cycle, why and when CDK1 change the role in cell
cycle progression. Here we show that apoptosis of Molt-4 cells (or
peripheral blood lymphocyte) in G1 phase of the cell cycle, which
occurs following DNA damage by ionizing radiation, coincides with
unscheduled activation of CDK1 in G1 and CDK1 was activated in G1
by unscheduled cyclin B1 accumulation, The data suggest that it is
possible that wrong time and wrong place of cyclin B1 accumulation,
which induce wrong time and wrong place of CDK1 activation, take a
role in apoptosis initial time and curial point.
173/P25
MEMORY EFFECTS OF THE G1-PHASE CELL CYCLE
CHECKPOINTS
Yixin Tong1, Daxing Xie1, Deding Tao1, Junbo Hu1, Jianping
Gong1
1
The cell cycle phase-specificity of apoptosis induced by X-ray was
recognized as the outcome of cell cycle checkpoint mechanisms. In our
previous study, we have demonstrated that low dosage of X-ray (2Gy)
treatment of Molt-4 cells resulted in transient G2-phase arrest and G1phase
apoptosis(63.97%).
Roscovitine
[2-(R)-(1-ethyl-2hydroxyethylamino)-6-benzylamino-9-isopropylpurine] Ca potent
selective inhibitor of the Cdk1 C can effectively block X-ray induced
apoptosis (53.16%). The survivals of the cultured cells whose DNA
were damaged and checkpoint was dulled continued to proliferate and
pass through next cell cycle sequentially. Whether these survived cells
show the same response to repeated X-ray is unknown. To investigate
the memory effect of cell cycle checkpoint, in this study, we cultured
the survived cells for additional 10 to 14 days. When apoptosis reduced
to 12.1%, survival cells were exposed to X-ray again. Our results revealed
that even being irradiated for the second time, less cells went to apoptosis
than control group. These findings indicated that survived cells whose
checkpoint had been interfered by Roscovitine may had the potency to
tolerate repeated irradiation damage, which suggests that to some extent
cell cycle DNA damage checkpoint has a memory effect.
174/P26
CELL CYCLE SPECIFICITY OF APOPTOSIS IN
DIFFERENT COMBINATION TREATMENTS IN MOLT-4
CELL LINE
Yongdong Feng1, Chunzhao Yu1, Deding Tao1, Jianhong
Wu1, Jianping Gong1
1
Objective To study the apoptosis pattern in MOLT-4 cells induced by
combination treatment of chemotheraputics. Methods X-ray (10Gy),
JinKe, CPT, Ara-C, Vim-26, Vinblastine alone or in combination added
into a flask of MOLT-4 cells in log phase growth, giving a final
138
concentration of 3.0 g/L JinKe, 0.25 µmol/L CPT, 1.0 µmol/L Ara-C,
0.5 µg/ml Vim-26, 0.05 µg/ml Vinblastine. The incubation was performed
on MOLT-4 cells that were separately treated at the different times. The
flow cytometric method of API was used to quantitate apoptotic cells as
well as to analyze the specific phase of apoptotic cells in the cell cycle.
Results 3.0 g/L JinKe and 10 Gy X-ray, 0.5 µg/ml Vim-26 and 0.05 µg/
ml Vinblastine or CPT and Ara-C could augment the effect of each other
and increase the amount of apoptotic cells. A synergistic interaction
between the two drugs was found. Most apoptotic cells were still observed
in G1, S or G2/M phase of the cell cycle and the cell cycle specificity of
apoptosis was not changed. An additive interaction between JinKe and
CPT was also found, most apoptotic cells were still mainly observed in
G1 and S phase of the cell cycle. Another additive interaction between
JinKe and VM-26 was also found, and most apoptotic cells were observed
in all phases of the cell cycle. There was an additive interaction between
Vinblastine and CPT, most apoptotic cells were observed in S phase of
the cell cycle and a few were observed in G2/M phase. Conclusion
Combination treatment of drugs which effect in the same cell cycle
phase bring about a synergistic action, and combination treatment of
drugs which effect in different phases of cell cycle bring about a additive
action in MOLT-4 cell line.
175/P27
EFFECTS OF CAMPTOTHECIN AND CYTOSINE
ARABINOSIDE ON CELL CYCLE SPECIFICITY OF
APOPTOTIC CELLS DURING COMBINATION
TREATMENTS IN MOLT-4 CELL LINES IN VITRO
Deding Tao1, Chunzhao Yu1, Hui Xiao1, Xiaolan Li1,
Jianhong Wu1, Jianping Gong1
1
Objective To determine whether Camptothecin (CPT) could augment
Cytosine arabinoside (Ara-C) activity and could change the phase of
apoptosis in the cell cycle during combination treatments. Methods
CPT, Ara-C, alone or in combination were added to a flask of Molt-4
cells in log phase growth, giving a final concentration of CPT (0.25
µM), Ara-C (1.0 µM) or CPT (0.25 µM) + Ara-C(1.0 µM). Incubation
with Ara-C or CPT were performed on MOLT-4 cells that were treated
for 4 h to observe cellular apoptotic shapes with confocal laser scanning
microscopy. Furthermore, three different flow cytometric methods,
including SubG1, API Assay and Cyclin E/DNA, were used to analyze
the cell cycle specificity of apoptosis and expression of Cyclin E. For
further analysis, express of Bcl-2 were assayed by western blot . Results
CPT could augment Ara-C activity and increase the amount of apoptotic
cells. A synergistic interaction in the two drugs was found. Most apoptotic
cells were observed in S phase of the cell cycle. Expression of Cyclin E
had been elevated and expression of Bcl-2 had been decreased.
Conclusion There is a synergistic interaction in the two drugs
combinations in S phase of the cell cycle. The interactions still occur at
the same checkpoint in the cell cycle. Both Cyclin E and Bcl-2 play a
critical role in response to chemotherapy agents and checkpoint status
has been implicated in cell sensitivity to these drugs.
176/P28
COMPARISON OF CALCEIN-AM AND ANNEXIN-V AS
EARLY VITAL MARKERS OF APOPTOSIS BY
CYTOMETRY
Yongdong Feng1, Jia Wang1, Xiaolan Li1, Hui Xiao1,
Jianping Gong1
1
Objective The current study was designed to compare the sensitivity of
Calcein-AM and Annexin V-FITC for early detection of apoptosis in
living cells by Flow Cytometry and to estabilish a new method for early
detection of apoptosis. Methods The Molt-4 cell line treated with
camptothecin(CPT) or ultraviolet(UV) were labeled with Calcein-AM
and FITC-conjugated Annexin V respectively, then analyzed the
apoptosis rate by flow cytometry and compared the performance of
Calcein-AM and Annexin V for early detection of apoptosis. Results
Our results show that both probes allowed the detection of apoptotic
cells. However, Calcein-AM was more sensitive than Annexin V. We
could detect the apoptosis induced by CPT two hours after inducing
with Calcein-AM but three hours after inducing with Annexin V. And,
the apoptosis induced by UV could be detected one hours after inducing
with Calcein-AM but two hours after inducing with Annexin V.
Conclusion Annexin V is less sensitive than Calcein-AM for early
apoptosis detection, and the new method detecting early apoptosis with
Calcein-Am is stable, reliable, sensitive and low-cost
177/P29
FAS EXPRESSION IN G1 PHASE WAS CORRELATED TO
CELL CYCLE SPECIFIC APOPTOSIS IN PHA
STIMULATED PBL CELLS
Jing Hu1, Yongdong Feng1, Daxing Xie1, Deding Tao1,
Jianping Gong1
1
Objective To study the expression and its cell cycle specificity of Fas on
normal and phytohemagglutinin (PHA) stimulated peripheral blood
lymphocytes. The correlation between Fas expression and cell cycle
specific apoptosis was determined. Methods PBL was stimulated to
proliferate by PHA. Apoptosis was analyzed by API method. Expression
of Fas and its cell cycle specificity were analyzed by a developed doubleparameter flow cytometry. Apoptisis was induced by adding Fas ligand
(FasL) to log growing PBL cells. Results Fas expression on PHA
stimulated PBL increased by 33.62% as compared to normal PBL. Mostly,
the expression of Fas was in G1 phase. And, FasL induced apoptosis was
mainly in G1 phase. Conclusion Expression of Fas increased in PBL
cells when stimulated by PHA. G1 phase specific expression of Fas was
correlated to G1 phase specific apoptosis, applying that Fas expression
was cell cycle specific and play an important role in cell cycle specific
apoptosis.
178/P30
APOPTOSIS AND PROLIFERATION OF ORAL MUCOSAL
EPITHELIA CELLS AFFECTED BY THE NUTRITIONAL
STATUS
Xuelai Luo1, Deding Tao1, Chuanyong Yang1, Junbo Hu1,
Jianping Gong1
1
Introduction: The nutrition absorbed by body will be supplied to cells
for metabolism. So the shortage of nutrition will affect cells and the rate
of cell apoptosis and proliferation may be changed. Changes in nutritional
status may be reflected rapidly in fast proliferating cells, such as the
cells of oral mucosal epithelium (OME). The surface layer of OME has
been calculated to be replaced every 2.7h and they could be collected
easily. This fact coupled with the easy and non-invasive accessibility of
the OME for cytologic study purposes led us to test a hypothesis that
OME cell apoptosis and proliferation might correlate with nutritional
status. Research Methods & Procedures: Forty-two consecutive patients
(13 male, 29 female; mean age 54.3±11.8 years, mean body mass index
21.8±2.8 kg/m2) with gastrointestinal malignant tumors were
prospectively studied. Patient-Generated Subjective Global Assessment
was used to identify malnourished patients. Anthropometric measures
including weight, body mass index, triceps skinfold thickness and
midarm muscle circumference were recorded. The serum proteins
measured were retinol-binding protein, transferrin, prealbumin and
albumin. Simultaneously, the rates of oral epithelial cell apoptosis and
proliferation were measured by flow cytometry. Of the 20 malnourished
patients, 14 were followed up in a serial study with a 3 day nutrition
support therapy. Nutritional indices and oral epithelial cell apoptosis
rate were measured after 3 days of nutrition support. Results: Malnutrition
was diagnosed in 20/42 patients. Oral epithelial apoptosis and
proliferation rates were significantly decreased (p<0.001 and p<0.05
respectively) in malnourished as against non-malnourished patients
although there were no significant differences between their
anthropometric data. Compared with non-malnourished patients,
malnourished patients had lower serum levels of retinol-binding protein,
transferrin, prealbumin and rates of oral epithelial cell apoptosis and
proliferation. The rate of oral epithelial cell apoptosis positively
correlated with serum retinol-binding protein (R=0.32, p<0.05) and
serum prealbumin (R=0.33, p<0.05). The rate of oral epithelial cell
apoptosis, serum RBP and serum PA increased significantly after a 3day nutritional support in malnourished patients. Conclusions:
Malnutrition affected proliferation and apoptosis of human cells in
139
vivo. A significant correlation was found between oral epithelial call
apoptosis rate and nutritional status of surgical patients with
gastrointestinal malignant tumors. This could potentially be a new
addition in the array of nutritional assessment tools used in clinical and
research settings.
179/P31
NOVEL VIOLET-EXCITED REAGENTS FOR DETECTION
OF VIABILITY AND VITALITY
Gayle Marie Buller1, Jolene A. Bradford2, Stephen Yue3,
Jixiang Liu3, William L. Godfrey2
was only significant for the CD4+ and CD28+ populations. Similar data
were obtained in rat lymphocytes. CONCLUSIONS: The AR was more
sensitive apoptosis indicator than AI quantifying apoptosis in murine
cell cultures. This reflects that the AR measures with more accuracy than
the AI the real ocurrente of apoptosis, because AR takes into account
both the late apoptotic cells that divide into apoptotic bodies and the
apoptotic cells that loss their lineage antigen. (Table 1: p<0.05 Wilcoxon
Matched pairs signed-ranks test).
Apoptotic Rate (AR) vs Apoptotic Index (AI) in the quantification
of apoptotic cells in mouse cell cultures
C
D
3+
1
Invitrogen, Eugene, Oregon; 2Molecular Probes, Inc., Eugene,
Oregon; 3Molecular Probes, Inc, Chemistry, Eugene, Oregon
As violet diode lasers become more prevalent secondary excitation
sources on flow cytometers, there is the opportunity to move common
applications from the 488 nm excitation line to violet excitation. Viability
(membrane integrity) and vitality (enzyme activity) are common
applications that typically are performed using the green and red emission
channels off the 488 nm laser. We have developed several novel violetexciting organic dyes that can identify stressed or dead cells in stained
populations without sacrificing channels used for common fluorophores
such as Alexa Fluor ® 488, R-phycoerythrin (R-PE) and R-PE tandem
dyes. Cell TraceTM calcein violet, AM dye is a metabolic probe that can
indicate the level of intracellular esterase activity of a live cell as
determined by the enzymatic conversion of the nonfluorescent, cellpermeant acetoxymethyl ester (AM) to an intensely fluorescent violetexcited dye that is well-retained in the cell and emits around 440 nm.
Cell TraceTM calcein violet, AM staining is comparable to calcein, AM,
which is commonly used to assess vitality in flow cytometry and
microscopy. The vitality reagent can be used concurrently with annexin
V Alexa Fluor®488 and propidium iodide to add a measure of enzymatic
activity to the study of apoptosis. For viability measures, two violetexcitable dead cell stains are available that withstand aldehyde fixation
and have peak emission around 450 and 515 nm, respectively. These
amine reactive fluorescent dyes have the ability to covalently label
cells: dead cells label more brightly than live cells because the dye stains
the cytoplasm of cells that have lost membrane integrity. These dyes
stain equivalent dead cell populations versus ethidium monoazide
bromide (EMA), but they do not require an additional UV photolysis
step to cross-link EMA to the DNA of dead cells. The fixable dye with
peak emission around 515 nm can easily be combined with Cell Trace
calcein violet, AM to create a robust violet-excited live/dead assay.
180/P32
APOPTOTIC RATE VS APOPTOTIC INDEX IN THE
QUANTIFICATION OF APOPTOTIC CELLS IN BOTH RAT
AND MOUSE CELL CULTURES
Julio Chevarria1, Luis Chara1, David Diaz1, Alfredo Prieto1,
Jorge Monserrat1, Hugo Barcenilla1, Miguel A. Sánchez1,
Leonardo Acuña1, Norman Muñoz1, Melchor Alvarez De
Mom1
1
Alcala University, Immune System Diseases and Oncology
Madrid, Spain
BACKGROUND: Currently the apoptosis in culture is quantified by
the Apoptotic Index (AI), or the proportion of apoptotic cells. The
apoptotic Rate (AR), a new flow cytometry-based ratiometric method
extends the AI and provides a more accurately estimation of apoptotic
cells from several human lymphocyte subsets. OBJECTIVES: To
compare the sensitivity of AR vs AI in the quantification of apoptosis in
murine cell cultures. METHODS: Highly purified spleen lymphocytes
populations were obtained by positive fluorescence-activated cell sorting
(FACSaria) from Wistar rats and C57/BL6 mice. The cells were cultured
for 24 hours under four conditions: without exogenous apoptosis
inducers and in the presence of phytohemagglutinin (PHA), staurosporin
(ST) or polystyrene beads coated with CD3 and CD28 antibodies (T-cell
expander). We used a flow cytometry-based ratiometric method that
uses an internal reference estandar of microbeads combined with
antibodies, Annexin V-FITC binding and 7-Aminoactinomycin D to
enumerate viable, necrotic and apoptotic cells. Differences were evaluated
by the Wilcoxon test and were considered significant when p<0.05.
RESULTS: Significant differences were observed in all celular
populations in all culture conditions assayed, but in staurosporin that
140
Sponta
neous
PHA
ST
TCE
C
D
4+
C
D
8+
C
D
19 +
C
D
5+
C
D
28 +
AR
AI
AR AI AR AI
AR AI AR AI
AR AI
0.50*
0.31
0.47* 0.32 0.48* 0.28
0.86* 0.78 0.55* 0.39
0.47* 0.32
0.60*
0.37
0.46* 0.32 0.60* 0.36
0.89* 0.81 0.59* 0.41
0.46* 0.32
0.95
0.93 0.74* 0.63 0.98 0.97 0.97 0.94 0.89 0.84 0.74* 0.63
0.48*
0.31
0.58* 0.32 0.45* 0.28
0.89* 0.81 0.56* 0.36
0.58* 0.32
181/P33
LATE APOPTOTIC CHANGES IN CHROMATIN
STRUCTURE AND DNA CONTENT DETECTED WITH
VYBRANT DYECYCLE STAINS
Jolene A. Bradford1, William L. Godfrey1
1
Molecular Probes, Inc., Flow Cytometry, Eugene, Oregon
Apoptosis is a carefully regulated process of cell death that occurs as a
normal part of development, a cascade of events leading to complete
cell deconstruction. These changes occur through any of several well
characterized pathways, including cell surface signaling, changes in
morphology, and caspase activation, all resulting in DNA condensation
and fragmentation, and eventual loss of plasma membrane integrity.
We have found that the new cell-permeant DNA stains, Vybrant®
DyeCycle™ violet and Vybrant® DyeCycle™ orange stains, detect
changes in DNA structure associated with apoptosis using 405 nm and
488 nm excitation, respectively. The UV-excited dye, Hoechst 33342,
is known to stain condensed chromatin of apoptotic cells more brightly
than non-apoptotic cells, a response also seen with Vybrant® DyeCycle™
violet stain using 405 nm excitation. The violet-excited dye was used in
a six-hour time course of camptothecin-induced Jurkat cells co-stained
with 7-AAD as a dead cell discriminator. Cells could be classified into
three categories: viable, apoptotic and dead. Apoptotic cells with
condensed chromatin initially appeared around three hours postinduction and their prevalence increased with further time of induction.
DNA fragmentation is a later apoptotic event which follows nuclear
compaction, and is characterized by the appearance of a sub-G0 peak in
apoptotic cells. Using the cell-permeant Vybrant® DyeCycle™ orange
stain in dual staining with SYTOX® Blue dead cell discriminator, a six
hour time course using camptothecin-induced Jurkat cells was
performed. Dead cells were gated out, leaving only live cells to be
analyzed for cell cycle. The sub-G0 population first appears around 34 hours post induction and increases with additional time of induction.
Further characterization of the sub-G0 population was done using a sixhour time course of camptothecin-induced Jurkat cells stained in three
colors using the monomeric cyanin, PO-PRO™-1 iodide (an apoptotic
marker similar to annexin V), a red-excited dead cell discriminator, and
Vybrant® DyeCycle™ orange stain. Progression of cells through a
sequence is demonstrated showing live progressing to early apoptotic
events, then to late apoptotic events, and finally progressing to dead
cells.
182/P34
SIX-COLOR CYTOMETRIC ANALYSIS OF FAS/FASL
REGULATION OF MURINE BASOPHILIC
ERYTHROBLAST HOMEOSTASIS
Renold J. Capocasale1, Dorie Makropoulos1, Amy Volk1,
Jeffrey Arlen1, John Quinn2, Ram Achuthanandam1, Peter
Bugelski3
1
Centocor, Inc., Radnor, Pennsylvania; 2Drexel University,
Biomedical Engineering, School of Biomedical Engineering,
Science, and Health Systems, Philadelphia, Pennsylvania;
3
Centocor, Toxicology and Investigational Pharmacology/
Experimental Pathology, Malvern, Pennsylvania
Many studies have shown Fas-Fas Ligand (FasL) mediated apoptosis to
be important in maturation and differentiation of erythroid precursors
in vitro. To determine if there is a similar process regulating
erythropoietic homeostasis in vivo, we studied erythropoiesis in Fas(lpr)
and FasL (gld) deficient mice. We postulated that deficiency of Fas or
FasL should result in changes in red blood cell (RBC) parameters and/or
decreased levels of apoptosis of erythroblasts. To test this hypothesis
under steady state conditions, blood and bone marrow were collected
from 10-week old C57Bl/6, B6.MRL-Tnfrsf6 lpr /J CD95 deficient
mice, and B6Smn.C3-Tnfsf6 gld /J CD95L deficient mice. Hematology
was studied using a Bayer Advia 120 and femoral bone marrow was
analyzed by 6-color flow cytometry using a Becton Dickinson FACSAria.
Hematologic analysis revealed no differences in reticulocyte counts,
RBC counts or hemoglobin (Hgb) in either lpr or gld mice compared to
C57Bl/6 controls. Similarly, analysis of bone marrow revealed no
differences in % of basophilic erythroblast (BEB, Ter-119 bright CD71
bright), % apoptotic BEB (annexin V+, 7-AADdim) or % FasL+ BEB in
either gld or lpr mice compared to control. As expected, lpr mice
expressed 10 fold fewer Fas+ BEB while similar levels were observed in
gld mice compared to controls. To test our hypothesis under stimulated
conditions, control, lpr and gld mice received a single s.c. dose of
10,000 units of recombinat human erythropoietin (rhEPO). Bone
marrow samples were collected 48 hours after dosing and blood samples
4, 8 and 16 days after dosing. Hematologic analysis revealed no
differences in the erythropoietic response among the three strains of
mice tested. Moreover, treatment with rhEPO had no effect on % Fas+
BEB in any strain, but induced a 2-5 fold increase in the % FasL+ BEB
and a 2-3 fold increase in apoptotic BEB in all three strains. Based on
our observations, we conclude Fas/FasL is unlikely to play a pivotal
role in regulating erythroid homeostasis.
183/P35
CHARACTERIZATION OF AGONISTIC CD28 SIGNALING
IN JURKAT AND H9 T CELLS
Mary A. Turner1, Michael McDermott2, Ashvin Dewan2,
Ashley Martin2, John Rodgers2, Dorothy E. Lewis2
1
Baylor College of Medicine, Immunology, Houston, Texas; 2Baylor
College of Medicine, Immunology, Houston, Texas
CD28, a key co-stimulatory molecule on T cells, is important for
optimal T cell activation. Until recently, it was thought that stimulation
with CD28 enhanced T cell receptor (TCR) signaling but had no effect
on its own. However, certain CD28 monoclonal antibodies (mAb) act
in a superagonistic manner, independent of stimulation via the TCR.
Possible mechanisms for this superagonistic reactivity may include
differences in calcium mobilization, CD28 lipid raft co-localization,
Granzyme B upregulation, and NF-kB nuclear translocation. Agonistic
CD28 mAb (ANC28) induces both activation (CD69 upregulation) and
apoptosis (AnnexinV binding) of the T cell line, Jurkat. Another T cell
line, H9, while activated, does not demonstrate apoptosis. A conventional
CD28 antibody did not induce apoptosis or activation in either cell line.
NF-kB components p65 (RelA) and c-Rel were translocated after
sustained ANC28 treatment. In Jurkat T cells, c-Rel was induced after
15 min and sustained for at least 90 min. The reverse was true in H9,
p65 (RelA) was induced after 15 min and sustained for at least 90 min.
To examine early responses to ANC, we analyzed calcium flux using
Fluo3/Fura 2. In Jurkat T cells, flux was delayed in ANC28-treated cells
compared to ionomycin or anti-CD3 treated cells. H9 T cells demonstrated
normal calcium flux with ionomycin and anti-CD3, but did not respond
to ANC28. Because T cell activation is associated with redistribution of
T cell surface molecules, we examined co-localization of lipid rafts
after ANC treatment in Jurkat and H9 T cells using cholera toxin as a
marker of lipid rafts. CD28 co-localization with lipid rafts occurred in
Jurkat, but not H9 T cells. In addition, CD28 positive apoptotic bodies
were observed in Jurkat, but not H9 T cells after ANC28 treatment.
Microarray analysis identified upregulation of several NF-ÛB related
genes (IkBA, c-Rel, NF-kB1, and Rel B) as well as Granzyme B induced
by ANC28 in Jurkat, but not H9 cells.
These data suggest that ANC28
signaling differences between Jurkat and H9 T cells begin with
differences in calcium flux, CD28 co-localization, followed by NF-ÛB
and Granzyme B upregulation. These differences are implicated in the
apoptotic response observed in Jurkat T cells, but that is not observed in
H9 T cells.
184/P36
LOSS OF LINEAGE ANTIGENS IN RAT AND MOUSE
APOPTOTIC LYMPHOCYTES
Luis E. Chara Velarde1, Julio Chevarria1, David Diaz1,
Alfredo Prieto1, Jorge Monserrat1, Hugo Barcenilla1, Miguel
A. Sanchez1, Leonardo Acuna1, Norman Munoz1, Melchor
Alvarez-Mon1
1
Laboratory, CNB-CSIC R&D Associated Unit, University of Alcala,
Alcala de Henares, Madrid, Spain
BACKGROUND: Recently, it has been demostrated that the loss of
Lineage Antigens (LAg) expression appeared to be a common feature
of apoptotic lymphocytes in humans, but this phenomenon had not
been studied in murine models. OBJECTIVES: To determine if the loss
of LAg expression is a common feature of rat and mouse apoptotic
lymphocytes, and to study the kinetic patterns of their LAg expression
along the apoptotic process. METHODS: Highly purified spleen
lymphocyte populations were obtained by positive cell sorting from
Wistar rats and C57/BL6 mice. The cells were cultured for 24 hours
under 4 conditions: without exogenous apoptosis inducers and in the
presence of phytohemagglutinin(PHA), staurosporin(ST) or T-cell
expander(TCE). We used a 4 color flow cytometry analisys with surface
antibodies, Annexin V binding and 7-Aminoactinomycin D to enumerate
viable (V), necrotic, and early (EA), intermediate (IA) and late (LA)
apoptotic cells. Differences were evaluated by the Wilcoxon test, p<0.05.
RESULTS: We observed loss of all antigens studied in apoptotic cells
from both species rat and mouse. A progressive decrease of the Mean
Fluorescence Intensity (MFI) in the diferent studied LAgs along the
apoptosis process was observed in all lymphocyte populations in
spontaneous and induced apoptosis. The kinetic of LAg loss was faster
for the CD28 and CD19 antigens in the early apoptosis stages, and was
progressive along the different apoptosis stages for the CD4, CD8 and
CD3 antigens. In the case of CD5, the LAg expression was maintained
in the early apoptosis stages and decreased in the intermediate and late
apoptosis stages. The percentage of final MFI LAg loss in respect to
viable lymphocytes was more than 90 % for the CD28 LAg, between
50% and 90% for the CD5, CD8 and CD19 LAgs, and between 20% and
50% for the CD3 and CD4 LAgs. CONCLUSIONS: Under all the
conditions assayed, the loss of LAg expression is a common feature of
rat and mice apoptotic lymphocytes. The loss of antigen by apoptotic
lymphocytes it has been demostrated in the three different species:
human, mouse and rat; this suggest that the loss of antigens by apoptotic
lymphocytes is a common feature of mamalian species. The different
kinetic patterns of LAg loss observed for different surface proteins
suggest that this process might be actively regulated by apoptotic cells.
Kinetic of % loss of LAg expression in mouse lymphocytes
undergoing Spontaneous (A) or PHA (B), ST (C) or TCE (D)
induced apoptosis
141
185/P37
EFFECTS OF HEAT SHOCK PROTEIN 72 ON
NEUTROPHIL FUNCTION
Andrew Osterburg1, Sandy Schwemberger1, George F.
Babcock2
1
Shriners Hospitals for Children, Research, Cincinnati, Ohio;
2
University of Cincinnati, Surgery, College of Medicine, Cincinnati,
Ohio
Heat shock proteins (HSP) play an important role in the regulation of
the immune response following severe traumatic injuries, such as burns.
We have previously reported that following severe thermal injury,
neutrophils (PMNs) display altered function including altered ability of
undergo apoptosis and upregulate CD11b. In this study, we examined
effect of HSP72 expression on nuclear factor kappa B (NF&kappaB),
apoptosis, CD11b in PMNs. HSP72 expression was induced in PMNs
obtained from healthy individuals by incubation at 41&degC for 60
min. and detected by flow cytometry. Apoptosis is induced by incubating
PMNs with 400 IU of TNF&alpha for 90 min. HSP72 expression, CD11b
expression, NF&kappaB, and apoptosis were detected by flow cytometry
following the binding of fluorochrome labeled antibodies to HSP72
(HSP70b´), CD11b, NF&kappaB or fluorochrome labeled annexin V
for apoptosis. Confocal microscopy studies, and in some cases
immunoprecipitation followed by Western blotting, were also performed
to detect CD11b and co-localization. Incubation at 41&degC induced
the expression of HSP72 in nearly 100% of the PMNs. Following
treatment with TNF&alpha, 30-45% of PMNs were apoptotic. In PMNs
expressing HSP72, apoptosis was reduced to <10%. The levels of the
active form of NF&kappaB (p65) increased in PMNs expressing HSP70.
Immunoprecipitation studies indicated that HSP72 and NF&kappaB
co-precipitated and, thus, may influence its translocation to the nucleus.
Co-localization to the nucleus was demonstrated by confocal microscopy.
The reduced up-regulation of the active epitope of CD11b correlated
with the level of expression of HSP72. The data is indicative of multiple
roles for HSP72, an anti-apoptotic role mediated through NKB, and an
alteration in upregulation of the active epitope of CD11b.
186/P38
OXIDATIVE STRESS PRODUCE CHANGES IN DNA
TOPOLOGY OF HUMAN LEUKOCYTES
Andrey Igorevich Poletaev1, Andrey Neustroev2, Maria
Vladimirovna Savvateeva1
1
Engelhardt Institute of Molecular Biology, Cytometry Unit,
Moscow, Russia; 2Federal State Institute 40-SSRI, Ministry of
Defense, Russian Federation, Immunology Lab., Lomonosov,
Russia
We designed the test, able to detect small number of DNA damages,
produced by oxidative stress. The test is based on detection of changes
in DNA staining ability by ethidium bromide (EB). Oxidative stress in
human leucocytes changes the topological state of nuclear DNA - average
number of super helix turns in chromatin domains. The number of
negative turns in the loops of chromatin DNA rules the binding ability
of well know intercalator EB. Fast staining of cells nuclea using NP-40
(0.2%) and staining buffer, containing Mg2+ and Ca2+, leads to activation
of chromatin associated nucleases, and produces numerous nicks in
chromatin loops. Such staining relaxes all super turns in DNA and
makes thus staining level independent on initial functional topology of
nuclear DNA. If one performs staining in the presence of nucleases
inhibitors, the final level of staining will be dependent on average
number of super helix turns in chromatin domains. The difference in
staining levels in these two cases normally is between 1.42 and 1.50 (R).
This parameter reflects the topological integrity of chromatin loops. It
was shown that different factors provoking oxidative stress in cells
decreases parameter R. Monitoring of R-value makes it possible to trace
both the kinetics of stress rise and the overall DNA repair and DNA
topology restitution. This simple technique has been proved to be simple,
fast, and useful in numerous experiments with human cells exposed to
hyperbaric or hyper oxidative conditions.
142
187/P39
KINETICS AND EXPRESSION OF CD59 EXPRESSION IN
CHO AL CELLS ALTERED WITH PHOSPHOLIPASE C
AND RNAI
Carley Ross1, Michael H Fox2
1
Colorado State University, Cell and Molecular Biology, Colorado
State University, Fort Collins, Colorado; 2Colorado State University,
Cytomation GTX, Cell and Molecular Biology, Department of
Environmental and Radiological Health Sciences, Fort Collins,
Colorado
The expression of the human cell surface antigen, CD59, on Chinese
hamster ovary A L cells is well characterized as an effective tool to
measure mutations in human chromosomes using the Flow Cytometry
Mutation Assay (FCMA Ross 2005, Zhou 2005). Temporal differences
are seen in the CD59 mutant expression between different mutagens,
such as lead acetate and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).
This observation led us to analyze the kinetics of expression and loss of
CD59 on the cell surface. CD59 is attached to the plasma membrane
through a glycosylphosphatidyl inositol (GPI) linkage. We analyzed
the minimum time required for CD59 to be expressed after cleavage
with phosphatidyl inositol phospholipase C (PI-PLC) and for protein
expression to cease when silencing the CD59 mRNA with RNAi. After
staining wild-type cells with directly conjugated fluorescent monoclonal
antibody to CD59, we measured the temporal protein expression on a
Dako CyAn flow cytometer. We found that the CD59 expression
decreased to 10% of the control after 1 hr treatment with PI-PLC and
fully recovered after 8 hours. We then silenced the CD59 mRNA using
Dharmacon Smart Pool siRNA for CD59. The protein expression
decreased to background levels beginning at 5 hr post treatment and
continued to about 15% of the control expression at 48 hrs. Both of
these experiments together suggest that treatment with a mutagen
affecting either the GPI-linked expression or the mRNA synthesis of
CD59 would take, one to two days before loss of CD59 expression
could be measured. Since the earliest expression of mutants is about 6
days, other processes must be involved in regulating the loss of CD59
surface expression after mutagenesis.
188/P40
IDENTIFICATION OF SMALL SUBPOPULATION OF
CELLS FROM HUMAN ANAPLASTIC THYROID
CARCINOMA
Ping Zhang1, Hui Zuo2, Wan-Tao Chen3, Kennichi Kakudo2
1
Hospital,Shanghai Key Lab of Stomatology, Shanghai, 200011,
China; 2Wakayama Medical University, Department of Pathology,
Wakayama, 811-1, Japan; 3Shanghai Second Medical University
Affiliated Ninth People’s Hospital, Shanghai, China
Purpose: There is increasing evidence that cancer contains its own stem
cells which contribute to tumor progression and drug resistance in a
variety of malignancies. It has been successfully isolated from brain
tumors, leukemias, and breast carcinomas and generally simulates some
phenotypes of corresponding normal stem cells. Anaplastic thyroid
carcinoma is one the most fatal malignancy to human, characterized
with rapid growth, wide spread and poor or un-differentiation. In this
study, we tested the hypothesis that if anaplastic thyroid carcinoma
contains subpopulations of cells that express known stem cell markers.
Methods: Two anaplastic thyroid carcinoma cell lines were examined
for the presence and the positive rates of stem cell markers by
immunohistochemical staining, immunofluroscent staining, flow
cytometry and RT-PCR. The checked stem cell markers include some
well-recognized embryonic stem cell markers and some putative adult
stem cell markers. Results: Flow Cytometry analysis detected the presence
of subpopulations positive for stem cell markers Oct-4, SOX2, SSEA1,
SSEA4, PODXL, p63, AC133 and ABCG2 in anaplastic thyroid
carcinoma. Immunohistochemical staining and immunofluroscent
staining analysis further confirmed the presence of cells immunoreactive
to Oct-4, SSEA1, SSEA4, PODXL, p63 and SOX2. Conclusions:
Human anaplastic thyroid carcinoma contains a small subpopulation of
cells that exhibit stem cell markers. Though evidence still waits to ascertain
their cancer stem cell identity, these findings point out that heterogeneity
do exist in anaplastic thyroid carcinoma that may have significant impact
on future treatment strategies. Acknowledgement:The study was
supported by National Natural Science Foundation of China,Grant
No.30300388,30330580
189/P41
ADHERENT CELL DISSOCIATION FOR FLOW
CYTOMETRIC PHOSPHOPROTEIN ANALYSIS
Piet Van Erp1, Diana Olthuis1, Lisa Green2, Rita Bowers2,
Haiyan Long2, Philip Marder2
1
Radboud University Medical Centre, Dermatology, Nijmegen, ,
Netherlands; 2Lilly Corporate Ctr, Indianapolis, Indiana
Upon activation, skin derived epithelial cells express a plethora of
cytokines, chemokines and accessory molecules, which can transmit
both positive and negative signals to cells of innate and adaptive immunity.
Dysregulation and abnormal expression of inflammatory mediators or
their receptors in keratinocytes and their associated signaling cascades
are relevant to the pathogenesis of chronic inflammatory skin diseases
such as for example psoriasis and can be studied in vitro. Flow cytometric
phosphoprotein analysis is a promising novel technique for studying
these signaling pathways. In order to obtain single cell suspensions
from cell cultures, cell dissociation and detachment of the monolayer is
required. However, during this proteolytic digestion procedure the
activation state of the cells may change and subsequent measurements
may not reflect the state of the cells at the end of the experimental
incubation. The purpose of this study was to determine conditions of
fixation and dissociation of human keratinocyte monolayers that permit
a reliable flow cytometric determination of phosphoproteins.
Measurement of phorbol ester-stimulated phosphorylated ERK1/2
(pERK1/2) served as a model read out system. This method was
optimized from a whole blood pERK1/2 biomarker assay. Different
fixation, permeabilization and trypsinization conditions were tested and
the sequence of use was varied. Best cell recovery was obtained and cell
integrity was maintained when fixation of the monolayer in 1%
paraformaldehyde for 15 minutes was used prior to a short trypsin
treatment followed by 90% methanol permeabilization of the resulting
cell suspension. Phorbol ester activation kinetics in blood monocytes
and cultured keratinocytes was very similar. However blood monocytes
were at least 10 times more sensitive to the stimulus. The described
method permits the reliable determination of pERK1/2 in single cell
suspensions derived from formaldehyde-fixed keratinocyte monolayers
and may be used for studying signaling pathways involved in the
pathogenesis of chronic inflammatory skin diseases. Furthermore, the
general methodology described in this paper has the potential for wide
application to the study of signal transduction in single cells derived
from adherent cell cultures using flow cytometry.
190/P42
IMPROVED ANALYSIS OF INDIVIDUAL
MITOCHONDRIA BY HIGH SENSITIVITY FLOW
CYTOMETRY
James P. Freyer1, Claire Sanders1, Stephanie Field1, Robb
Habbersett1
1
Los Alamos National Laboratory, Bioscience Division, Los
Alamos, New Mexico
Analysis of individual mitochondria by flow cytometry has the potential
to address both basic and clinical research questions in a rapid, highthroughput manner. However, individual mitochondria are at the lower
end of the resolution scale of most commercial flow instruments, which
perhaps explains why relatively little is published in this area. We are
using a home-built high sensitivity (HS) flow instrument (Habbersett
and Jett, Cytometry 60A: 125, 2004), originally designed to measure
DNA fragments, in order to improve the measurement of light scatter
and fluorescence dye parameters of isolated mitochondria. Mitochondria
were isolated from two different human tumor cell lines known to have
differing numbers and sizes of mitochondria. Mitochondrial suspensions
were labeled with four different commercial mitochondrial-specific
fluorophores and then analyzed on a commercial flow sorter and on the
HS instrument. Light scatter and fluorescence signals from the
commercial instrument could distinguish the mitochondria from the
different cell lines. However, measurement of fluorescent dye uptake
kinetics and sorting of mitochondria demonstrated several problems
with the analysis of these organelles by conventional flow cytometry.
Fluorescence intensity as a function of time after adding dye showed
very strange kinetics, with maximum fluorescence being achieved as
fast as could be measured (~2 minutes) followed by a gradual decline
over the next 30 minutes to a steady-state level ~25% of the maximum.
Steady-state fluorescence intensity varied from 10-100 times greater
than the autofluorescence signal, depending on the cell of origin. As
has been reported previously, forward and side light scatter signals
showed very broad peaks, producing a tear-drop shaped distribution on
a bivariate log-log plot. When mitochondria were sorted from the lowand high-intensity light scatter regions and then reanalyzed, the resultant
light scatter distributions were indistinguishable from those of the unsorted
population. These unexpected results suggest several problems with
analysis of isolated mitochondria by conventional flow cytometry,
probably related to tumbling of these non-spherical organelles, a very
small particle volume relative to the probe volume, and a significant
probability of multiple mitochondria being detected as a single event.
The design of the HS instrument should address these limitations.
Preliminary analysis has demonstrated that we can measure light scatter
and fluorescence signals from individual mitochondria using the HS
instrument. We will report on comparison of mitochondrial measurements
using conventional and HS instrumentation. Supported by NIH grants
CA-108853 and RR-01315 (National Flow Cytometry Resource).
191/P43
MULTIPARAMETRIC FLOW CYTOMETRIC
EVALUATION OF CYTOTOXIC T LYMPHOCYTE
POPULATIONS
Julie G. Wilkinson1, Sybil S. D’Costa1, Enrique Rabellino1
1
Beckman Coulter, Inc., Custom Biopharma Solutions, Miami,
Florida
Cytotoxic T lymphocytes (CTL) are the principal cells of the acquired
immune response for defense against viral infections and tumors. The
accurate analysis of such a functional effector population is critical to
the elucidation of correlates of protection for diseases involving cellular
immunity. Once standardized and validated, these assays can be routinely
used as surrogate markers of efficacy in preventative and therapeutic
strategies involving immune response. Multiparametric flow cytometry
has enabled the accurate identification and evaluation of targeted
lymphocyte subpopulations. In the current study, the authors have
evaluated the functional capacity of effector T cells using 5 color, 7
parameter flow cytometry by interrogating a combination of phenotypic
and functional surface and intracellular markers. Peripheral blood
mononuclear cells obtained from healthy donors were subjected to
restricted polyclonal stimulation using Staphylococcal enteretoxin B or
peptide specific stimulation for varying times ranging from 6-72 hrs.
The T cell populations were analyzed using combinations of markers
differentiating naïve, effector, memory, activated and proliferating
subpopulations along with functional evaluation utilizing intracellular
cytokine, granzyme B, perforin and degranulation as assessed by
activation-induced CD107 expression. Our findings indicate that there
is a complex profile of effector T cells that varies with the donor and
time post stimulation. The functional capacity of effector T cells is
especially dependent on the “instinsic” state of the donor PBMC
population with granzyme B up-regulation being a striking feature of
the response. Such profiles when correlated with disease outcome could
enable the targeted identification of effector CTL subpopulations
associated with therapeutic success thus enabling the development of
“surrogate profiles” of efficacy.
192/P44
STUDY OF CYTOKINE PRODUCTION BY FOXP3
EXPRESSING CELLS BY 8 COLORS
MULTIPARAMETRIC FLOW CYTOMETRY
Manuel Leonardo Acuña1, Hugo Barcenilla1, Jorge
Monserrat1, Alfredo Prieto1, David Diaz1, Martin Villarroel1,
Angela Hernández1, Eduardo Reyes1, Luis Chara1, Julio
Chevarria1, Melchor Álvarez De Mon1
1
CNB-CSIC R&D Associated Unit, Department of Medicine,
University of Alcala, University of Alcala, Alcala de Henares,
Madrid, Spain
Regulatory T cells play a key role maintaining the homeostasis of the
immune system. They have been well defined as CD4+CD25high cells
that express the transcription factor Foxp3. However, in humans the
expression of Foxp3 is not exclusively confined to CD4 +CD25high
cells, a population of CD25low cells with naïve phenotype also expresses
Foxp3 at lower levels than CD25high cells. In addition, activated human
T cells are able to express Foxp3. Objetive: By the use of multiparameter
flow cyometry in eight colors we have studied the production of
cytokines by different subsets of T cells defined by the expression of
143
Foxp3. Materials and Methods: We have studied peripheral blood
mononuclear cells (PBMC) from 6 healthy controls. Freshly or activated
PBMC were cultured for six hours in the presence or the absence of
PMA (50 ng/ml) plus Ionomycin (1 µg/ml). Monensin (2 µM) was
added to the cultures to retain the produced cytokines within the producer
cells. For surface labeling, we used antibodies against the following
antigens: CD3, CD8, CD45RA, CD25 CD27 CD31 and for intracellular
labeling, antibodies against: TNFá, IFNã, IL-2, IL-4, IL-10 and Foxp3.
Results: We have found significant differences in the intracellular
production of TNFá, IFNã and IL-2 in the different subsets defined by
Foxp3 expression and also by their naïve or memory phenotype. Foxp3+
cells express reduced levels of most of the cytokines especially INFã
compared to the foxp3- cells Conclusion: The combination of
intracellular cytokines and foxp3 staining at the single cell level allow
to distinguish subsets of T cells with different immunoregulatory function.
193/P45
DERIVATION AND CHARACTERIZATION OF HES
CELLS LINES WITH CRE-DEPENDENT RNAI OF
KINESIN-1 SUBUNIT KLC1
Rhiannon L. Nolan1, Nikole Kimes1, Jessica Flippin1,
Lawrence S. Goldstein2
1
University of California, San Diego, La Jolla, California;
University of California, San Diego, Pharmacology, School of
Medicine, La Jolla, California
2
Recent work from our lab suggests that neurons of individuals affected
by Alzheimer’s disease are likely to contain axonal defects. These defects
are enhanced in mouse models expressing reduced levels of the
microtubule-based motor protein kinesin-1 (Stokin et al 2005). To model
these defects in human cells, we derived human embryonic stem (hES)
cell lines with regulated RNA interference against the kinesin-1 subunit
KLC1 (kinesin light chain 1). We transduced HUES9 hES cells with
lentivirus packaged to carry cre-dependent expression of a small hairpin
RNA sequence directed against KLC1. We then isolated and propagated
individual colonies containing infected cells. The resulting cell lines
produced colonies that appeared morphologically normal, differentiated
into cells of neuronal morphology similarly to the parental HUES9 line,
and were karyotypically stable. We are now using these cell lines to
reduce kinesin levels and to model cellular transport defects in human
neurons differentiated in vitro.
194/P47
RESTRICTION POINT DEFINITELY EXISTS IN
NORMAL LYMPHOCYTES AND IS BROKEN IN MOLT-4
LEUKEMIC CELLS
Jichao Qin1, Yongdong Feng1, Xiaolan Li1, Daxing Xie1, Hui
Xiao1, Deding Tao1, Junbo Hu1, Jianping Gong1
1
Huazhong University of Science and Technology, Cancer Research
Institute, Tongji Medical College, Wuhan, Hubei, China
Although the presence of restriction point in G1 phase of some
transformed cell lines was demonstrated three decades ago (1), and its
absence that leads to uncontrolled proliferation in cancer was taken for
granted (2), there are several shortcomings in research on restriction
point: 1) transformed cell lines were used, not true normal cells; 2) with
the cell synchronization methods that were applied severe bias occurs
due to growth imbalance (3); 3) only some synchronized cells, not the
whole cell population, was able to initiate DNA replication (1). So,
there was no evidence until now to directly prove existence of the
restriction point in asynchronous normal cells, and luck of such in
cancer. In the present study, we used peripheral human blood
lymphocytes to confirm existence of restriction point as model of
asynchronous normal cells, and analyzed the molecular basis of it. We
also used post-sorting western blotting (4) to elicit non-existence of the
restriction point in the T-cell leukemia Molt-4 cells. These results might
provide directions for cancer therapy that spare normal cells thereby
decreasing side effects of the treatment
144
195/P48
EXPRESSION OF CYCLINS AND CDKS IN NORMAL
PROLIFERATING BONE MARROW CELLS IN VIVO
Daxing Xie1, Jing Yao1, Deding Tao1, Junbo Hu1, Yongdong
Feng1, Jianping Gong1
1
The key regulators of core cell cycle machinery which include cyclins,
CDKs and CKIs, have been elucidated in unicellular organisms and
cultured mammalian cells. However, expression of these regulators in
human proliferating cells in vivo has seldom been investigated. In our
study, freshly isolated bone marrow cells from healthy people were
collected and cell cycle regulators were analyzed by flow cytometry
and western-blotting. We found that during cell cycle of in vivo marrow
cells G1 cyclins (D3, E) did not expressed, and G2 cyclins (A, B1)
expressed not only in S and G2/M phase but also in G1 phase with their
levels increased throughout cell cycle phases gradually. Confocal
microscopy indicated that in G1 phase, cyclin A and B1 accumulated
predominantly in the cytoplasm, then they translocated into nuclear
during S and G2/M phase. Co-Immunoprecipitation and kinase assay
revealed that in the presence of high level of P27kip, CDK2 had very
low activity in G1 and S phases, CDK4/6 had no activity at all, while
cdc2 which bind both cyclin A and cyclin B1 was unexpectedly active
from G1, S to G2/M phase. These findings firstly demonstrated in vivo
expression of cell cycle regulators in human physiological proliferating
cells, and we suggest that such fashion differ from what was predicted
using studies in cultured cells.
196/P49
EXPRESSION OF CYCLINS IN HIGH-DENSITY
CULTURED CELLS AND IN VIVO GROWING CELLS
Daxing Xie1, Deding Tao1, Jing Yao1, Junbo Hu1, Jianhong
Wu1, Xiaolan Li1, Jianping Gong1
1
Cyclins are key components of the core cell cycle machinery. It is
widely assumed that during mammalian cell cycle, four major cyclins,
including G1 cyclins (D, E) and mitotic cyclins (A, B1), are expressed
in an orderly scheduled pattern in exponentially cultured cells. However,
in high-density cultured cells and in vivo growing cells, these cyclins
might not be expressed in the same schedule. In this study, we investigated
the expression of cyclins by flow cytometry and western-blotting in
cultured MOLT-4 cells at high-density (1×107/ml), bone marrow cells
from leukemic patients and HepG2 tumor cells from naked mice after
inoculation, respectively. We found that the levels of cyclin D, E, A
decreased evidently in each cycle phase compared to exponential cells,
and cyclin B1 moderately reduced within G2/M phase even expressed
in G1-phase cells. This unscheduled cyclin B1 was localized in G1phase was mainly in the cytoplasm, and its level was positively correlated
with the rate of apoptosis. Moreover, we also got the similar results in
HepG2 tumor cells from immune-deprived mice and in leukemic cells
from leukemia patients. These findings firstly demonstrated a new pattern
of cyclin expression in high-density cultured cells and in vivo tumor
cells which might recapitulate the microenvironment of cells in a living
organism, and we suggest that high-density cultures be more actually to
mimic in vivo growing conditions for cell cycle research.
197/P51
DYNAMIC BINDING OF PCG PROTEINS DURING
DROSOPHILA DEVELOPMENT
Jasper Grendel1, Cornelia Fritsch1, Gabriella Ficz2, Rainer
Heintzmann3, Donna J. Arndt-Jovin1
1
Max Planck Institute for Biophysical Chemistry, Molecular
Biology, Goettingen, Germany; 2The Barbaham Institute, Laboratory
of Developmental Genetics and Imprinting, Cambridge, United
Kingdom; 3King’s College London, New Hunt’s House, Randall
Devision of Cell & Molecular Biophysics, London, United
Kingdom
Polycomb group (PcG) genes are essential genes in higher eukaryotes
responsible for the maintenance of the spatially distinct repression of
developmentally important regulators like the homeotic genes. Their
absence, as well as overexpression, causes transformations in the axial
organization of the body. Although protein complexes have been isolated
in vitro little is known about their stability or exact mechanism of
repression in vivo. Using fluorescence recovery after photobleaching
(FRAP), we determined the translational diffusion coefficients of two
GFP fusion proteins of the PcG proteins, Polycomb and Polyhomeotic,
as well as the binding equilibrium and dissociation constants and residence
times for complexes of the proteins in vivo at different developmental
stages in whole living Drosophila organisms and tissues4. Although we
were able to make a rather complete analysis of both the diffusion and
the complex formation for these proteins, the small nuclei of Drosophila
cells as well as the movement of chromatin within the nuclear volume
present technical difficulties. Recently, a new method, called rasterimaging-correlation- spectroscopy (RICS), has been developed 5 for
determining diffusion constants of biological molecules in living cells
that utilizes the temporal information encoded in CLSM data using time
correlation analysis of the fluorescence fluctuations (FCS). We are
utilizing this technique to analyze the diffusion and binding equilibria
of another GFP labeled PcG protein, extra sex combs, ESC, which is
part of the histone methylation complex that directs the PcG protein
repression complex binding to chromatin. We present these data as well
as compare the pros and cons of the FRAP and RICS methods as
demonstrated by measurements of Polycomb-GFP in both systems in
living embryos and larval tissues. 4 Ficz, G, Heintzmann, R, ArndtJovin, DJ. Development 132: 3963, 2005. 5 Digman, MA, Brown, CM,
Sengupta, P, Wiseman, PW, Horwitz, AR, Gratton, E. Biophys. J. 89:
1317, 2005.
198/P52
ANALYSIS OF UV INDUCED DNA DAMAGE
CHECKPOINT BY CYCLIN E/DNA MULTIPARAMETER
FLOW CYTOMETRY
Daxing Xie1, Jianhong Wu1, Yongdong Feng1, Xiaolan Li1,
1
[Abstract] Background & Objectives Eukaryotic cell cycle events
progress strictly in order which is controlled by the mechanism of
checkpoint. At present, most analysis of checkpoints used the cytometry
to detect cell cycle distribution, which is named DNA histogram method.
In current study, we designed to set up and evaluate a new method for
analyzing G1L (late G1 phase) DNA damage checkpoint by Cyclins/
DNA multiparameter cytometry . Methods MOLT-4 Cells after being
irradiated by UV(ultraviolet) were gathered at different time points and
were divided into two groups. In one group, the total cell number of
G0/G1 phase was calculated by Modifit software using DNA Histogram
method. In the other group, fluorescence intensity and threshold of
cyclin E, and the cell number of sub-G1 phase (G0, G1 early, G1 late)
were quantitatively analyzed by Cyclin E/DNA multiparameter method
respectively. Results The increasing of G0/G1 phase cells (12.6%)
analyzed by DNA histogram method was observed at 6th hour after
cells being irradiated by UV. In contrast, the cyclin E fluorescence
intensity of G1 late phase (G1L) cells changed quickly, which rised
from 295.1 (control) to 341.2 (15.6%) at the first hour, and increased
to 577.6 (95.7%) at 6th hour. Simultaneously, cyclin E threshold rised
from 2.0 (0h) to 5.4 (6h). Conclusions Cyclin E/DNA multiparameter
flow cytometry is more sensitive and precise than DNA Histogram
method for detecting the initiation of G1L phase arresting and apoptosis,
which provided a scientific and reliable method for analyzing cell cycle
checkpoint.
199/P53
STUDY ON CYCLIN B1 AND CYCLIN E GENE
EXPRESSION IN POST-SORTING CELLS BY
QUANTITATIVE REAL-TIME PCR TECHNIQUE
Hui Xiao1, Daxing Xie1, Xiaolan Li1, Yongdong Feng1,
1
Objective To study on cyclins gene expression during cell cycle phases
is very useful for understanding the cell cycle regulatory mechanism.
The recently developed quantitative real-time PCR has proven to be a
highly effective tool for quantifying the gene expression. The purpose
of present study is to detect the expression level of cyclinB1 and cyclinE
gene in different phases of cell cycle by using flow cytometry sorting in
combination with quantitative real-time RT-PCR, and to discover the
law of cyclin expression during cell cycle at the transcriptional level.
Methods The asynchronously growing Molt-4 cells were sorted into
three subgroups (G0/G1, S, G2/M phases) according to DNA content
by flow cytometry. Total RNA was harvested from the sorted cells for
reverse transcription, and quantitative real-time PCR was performed on
Roche LighteCycler for analyzing expression level of cyclinB1 and
cyclinE gene. Results The gene expression level of cyclinB1 and
cyclinE varied during the cell cycle. The levels of cyclinB1 mRNA in
G0/G1 cells was 1.21 ~106 copy/ul, in S phase 5.05 ~107copy/ul, in
G2/M phase 2.73 ~109 copy/ul , respectively; The levels of cyclinE
mRNA in G0/G1 cells was 1.23 ~109 copy/ul, in S cells 3.34 ~107copy/
ul, in G2/M cells 4.03 ~107 copy/ul, respectively. Conclusions Both
cyclinB1 and cyclinE gene were expressed in all different phases of cell
cycle of asynchronously growing Molt-4 cells, however, their expression
pattern was not similar. The expression level of cyclinB1 mRNA during
G2/M phase was significantly higher than G0/G1 phase, and the cyclinE
gene expression increased in G0/G1 cells to almost 30 times that present
in G2/M cells. The results showed post-sorting real-time fluorescent
quantitative PCR was a sensitive and reliable technique to quantify the
gene expression in different phases during cell cycle of asynchronously
growing cells.
200/P54
INITIAL TIME OF APOPTOSIS WAS DEPENDENT ON
CELL CYCLE PROGRESSION IN HUMAN PBL AND
LEUKEMIA CELL LINES
Yongdong Feng1, Jianhong Wu1, Junbo Hu1, Daxing Xie1,
Xiaolan Feng1, Jianping Gong1
1
Apoptosis and cell cycle are intertwined in cell life and death. Although
it has been found that chemical or physical damage on cells could
enforced at specified cell cycle phase, which is the groundwork of
chemo- or physio-therapies in cancer, it is still unknown why faster
growing tumor is more sensitive to chemo or physio-therapies, and it is
not clear whether or not damaged cells always die at specified cell cycle
phase, and if so, what is the regular pattern of cell cycle specific apoptosis?
And how apoptosis programs during cell cycle progression? Here we
use a newly founded method of API that detects cell cycle specified
apoptosis to illustrate the kinetic changes of apoptosis during in cell
cycle progression. We tested both intrinsic and extrinsic apoptosis
inducers on leukemia cell lines as of MOLT-4 and Jurkat, and cultured
peripheral blood lymphocyte. We found that apoptosis mostly took
place at specified cell cycle phase. MOLT-4 and Jurkat cell were sensitive
to apoptosis insults both of intrinsic as of X-ray, CPT, Chinese medicine
Q and extrinsic as of TNF or Fas pathway. X-ray damage lead to G1
phase apoptosis, CPT induced S phase and Chinese medicine Q induced
apoptosis as of G1~S pattern. PBL cells under different driving or
hindering force or not present different pattern of apoptosis. PBL went
to apoptosis when stimulated by PHA and more cells went apoptosis
under additional insults of CPT, X-ray and others, while unstimulated
PBL kept blunt to these insults. And more, cells went ahead with
proliferation when caffeine abolished the cell cycle checkpoint.
Hungering or confluent PBL was blunt to apoptosis. It is concluded that
apoptotic cell death is a cell cycle event, which include that most
apoptosis (if not all) were initialed in the particular phases of cell cycle,
cells would evade apoptosis when they were stayed G0 or not stopped
in cell cycle progression and apoptotic schedule was varied with cell
145
cycle engine changed. Coordination of proliferation and apoptosis (CAP)
emerged here to describe this phenomenon, offering the platform for
both cell cycle and apoptosis.
201/P55
FLOW CYTOMETRIC ANALYSIS OF CELL CYCLE
SPECIFIC DIFFERENTIATION OF HL-60 CELLS
Ruojian Wen1, Daxing Xie1, Peng Zhang1, Deding Tao1,
Jianping Gong1
1
It has been found that all-trans-retinoic acid(ATRA) and hexamethylene
bisacetamide(HMBA) can both induce HL-60 cells to terminal
differentiation. However, the relationship between differentiation and
cell cycle progression is still in controversy. In this study, we used
multiparameter flow cytometry to detected the cellular surface antigen
CD11b expression and DNA content simultaneously, by which we could
analyze the cell cycle specificity of HL-60 differentiation during cell
cycle progression. Our results indicated that after HL-60 cells were
induced for 72 hours by ATRA i1.0 uM j, the CD11b expression was
evidently increased from 1.65% to 16.68%, and the CD11b positive
cells were largely located in G0/G1 phase. After incubated with HMBA
i10 uM jfor 24 hours, the CD11b expression was also increased from
1.82% to 13.33%, however, the CD11b positive cells were mainly located
in G2/M phase. DNA histogram analysis revealed that ATRA blocked
the cell progression in G0/G1 phase and HMBA greatly retarded the
progression of G2/M phase. From these findings, we propose that HL60 cell can be induced to differentiation within different cell cycle
phases by various inducers.
202/P56
CULTURE OF CELLS FROM PLACENTA AND BONE
MARROW IN BFGF CONTAINING MEDIUM GIVE RISE
TO FRIZZLED-9 AND SSEA-4 EXPRESSING MSC WITH
MULTI-POTENTIAL DIFFERENTIATION CAPACITY
Hans-Jorg Buhring1, Venkata Lokesh Battula1, Andreas
Boehmler1, Sabrina Treml1, Petra Bareiss2, Ingrid Albert3,
Sigrid Hojak3, Hans Kiefer3, Lothar Just2, Thomas Skutella2
1
University of Tubingen, Internal Medicine II, Tubingen, Germany;
University of Tubingen, Institute for Anatomy, Tubingen, Germany;
3
m-phasys, Tubingen, Germany
2
Mesenchymal stem cells (MSC) are plastic-adherent cells with fibroblastlike morphology that can be differentiated into several cell types including
bone, cartilage, muscle, stromal cells, tendon and connective tissue.
Culture of primary cells from human BM and placenta on gelatinecoated plates in the presence of bFGF resulted in the formation of MSC
with a more immature phenotype compared to the conventionally
prepared counterpart. Apart from their expression of “classical”
mesenchymal markers like CD13, CD105, CD164, and CDCP1 (CD318),
they additionally expressed the embryonic stem cell markers SSEA-4
and oct-4, as well as the progenitor marker nestin and the wnt receptor
Frizzled-9 (FZD9). Culture of these cells on ultra-low adherent dishes
resulted in the formation of large cell aggregates that resembled embryoid
body-like structures (EBS). Further differentiation of EBS in insulin,
transferrin and selenite (ITS) containing medium resulted in the formation
of adherent cells with fibroblast-like morphology. When cultured under
appropriate conditions, these cells gave differentiated into functional
adipocytes and osteoblast-like cells (mesoderm), glucagon expressing
pancreatic islet-like cells (endoderm), as well as class III -tubulin
expressing neuron-like and GFAP expressing astrocyte-like cells
(ectoderm). Our results demonstrate that the new culture protocol is
suitable for the generation of MSC that express novel markers including
Frizzled-9 and SSEA-4 and show multi-lineage differentiation capacity.
146
203/P57
MULTIPLEXED CHARACTERIZATION AND
MONITORING OF CULTURED ADULT MESENCHYMAL
STEM CELLS
Shayne Boucher1, Kate Wagner1, Ferenc Boldog1
1
Invitrogen Co, GIBCO Cell Culture Systems R&D, Grand Island,
New York
Biomedical researchers are facing the challenging task of translating
basic research findings on mesenchymal stem cells (MSC) to developing
effective cell-based therapies. One gap area is the lack of advanced
characterization and monitoring tools for studying MSC. MSC are
classically defined as fibroblast-like cells from fresh bone marrow
aspirates that can attach to culture surfaces and expand for a limited
number of passages. Upon exposure to specific cocktails, multipotent
MSC can differentiate into at least three cell types - adipocytes, osteocytes
and chondrocytes. Phenotyping and monitoring MSC cultures have
long relied on qualitative visual observations and stain-based bioassays
to morphologically identify MSC and differentiated cell types. These
time-intensive and laborious methods have served as a stimulus for
identifying less time-consuming and more informative methodologies
that can be utilized in cell culture studies. We implemented a panel of
research tools that allowed us to extract informative characterization
and monitoring data from in vitro studies in a more rapid manner. To
measure cell proliferation, we utilized a robust, fluor-based nuclear dye
assay kit that can test many conditions in a high throughput fashion as
compared to the traditional low throughput system that tested only a
few conditions. To monitor cell health, we employed cell-permeant and
-impermeant fluor dyes to rapidly detect plasma membrane integrity,
cytoskeletal matrix, apoptosis and other organelle functions in both
fixed and live cell format; this approach provided more health status
data as compared to the limited output of dye exclusion viability assays.
To phenotype multipotent and differentiated MSC, we leveraged
antibody and molecular biomarker tools to unambiguously identify
lineage-committed MSC; this substitution reduced assay time from 21
days to as little as three days. To characterize MSC activity at the genomic
level, we conducted qRT-PCR studies to generate gene expression profiles
that followed key genes involved in regulation of critical signaling
molecules; diagnostic differences in gene expression levels could be
detected within 24 hours. Since these technologies are not mutually
exclusive, we have begun to integrate them in a multiplexed and high
throughput format. For a given study, we can measure cell number,
monitor cell health, detect dead cells, and identify cell types. Furthermore,
by analyzing MSC gene expression profiles, we can predict downstream
activity detected by cytological means. We are moving forward to utilize
these technologies in a multiwell plate-based format. This would result
in a user-friendly, information-rich system that provides us a deeper
insight into the signaling pathways that regulate MSC function.
204/P58
CELL CYCLE ANALYSIS USING MICROPLATE
CYTOMETRY: A COMPARISON OF LASER AND DYE
COMBINATIONS
Tristan Cope1, Christopher Lupton1, Jolene A. Bradford2, Jeff
Hung2, Wayne P. Bowen1
1
2
TTP LabTech Ltd., Melbourn, Hertfordshire, United Kingdom;
Invitrogen Corporation, Eugene, Oregon
The cell cycle represents one of the most fundamental and important
processes in eukaryotic cells, culminating in cell growth and division
into two daughter cells. Defects in cell cycle regulation are a characteristic
feature of tumour cells and mutations in the genes involved in controlling
the cell cycle are extremely common in cancer. Monitoring dysfunctional
cell cycle regulation is thus the focus of intense interest, since it provides
an opportunity to discover new targets for anti-cancer drugs and improved
therapeutics. Traditionally, cell cycle analysis has been performed using
flow cytometry which measure changes in DNA content following
staining with fluorescent dye. The main disadvantages of this technique
are low throughput, use of large number of cells and the inability to
analyse adherent cell lines in situ. To address such issues, we have
developed a cell cycle analysis method using an Acumen Explorer
fluorescence microplate cytometer, capable of reading an entire 384
well microplate in under 10 minutes. The method can perform such
analyses on cells in situ, markedly simplifying sample preparation. Cell
cycle analysis is typically performed on permeabilised or fixed cells
using a cell-impermeant nucleic acid stain, but is also possible using live
cells and a cell-permeant nucleic acid stain. For fixed cell protocols the
most commonly used DNA dye is propidium iodide. It has the advantage
of being excited by 488 nm light and can be used on both flow and
microplate cytometers. While the choices for fixed cell staining are
varied, there are only a few examples of useful cell-permeant nucleic
acid stains, including the Vybrant® DyeCycle™ reagents. In this study,
we compared the performance of Vybrant® DyeCycle™ Violet stain
and Vybrant® DyeCycle™ Green stain, excited by 405 nm and 488 nm
laser lines respectively, with propidium iodide on both flow and
microplate cytometers. The results demonstrated a high degree of
correlation between the different DNA stains and cytometers, supporting
their integration in automated cell cycle protocols.
205/P59
IMPROVED METHODS FOR PLATELET ANALYSIS BY
FACS AND FOR STABILIZATION OF CD42B
EXPRESSION ON CORD BLOOD CULTURE-DERIVED
PLATELETS
Lucie Boyer1, Nicolas Pineault1
1
Héma-Québec, R&D, Quebec, Quebec, Canada
Due to the critical role of platelets (Plt) in hemostasis, reduction of
blood Plt counts requires the transfusion of Plts to safe levels. Our
laboratory focuses on the development of culture conditions for the
production of Plts from cord blood (CB) stem cells as a alternative
source to donated blood. Critical to our research, is the capacity to
enumerate and characterize the Plts produced in vitro in an easy and
reliable manner, and flow cytometry provides the best way to achieve
these objectives. The method used to assess Plt numbers is based in part
on work done by Norol et al. in 1998. In short, we first assess the cell
density (c.d.) of each cultures, then the Plts and cells are analyzed by
FACS using 2 analytical gates that are drawn based on the forward
(FSC) and side scatter (SSC) properties of normal blood Plts and cultured
cells. The Plt region excludes small microparticles and contaminating
cells. Only Plts co-expressing CD41 (GPIIb) and CD42 (GPIba)
(%CD41 +CD42+Plt), two antigens commonly used to characterize MK
and Plts, are considered true Plts. The relative proportions of Plts (%Plt)
and cells (%cell) in culture is also determined with the FACS analysis.
The density of Plts is then calculated as follow: c.d. X (%Plt / %cell) X
% CD41 +CD42+Plt. Although this method is simple and reproducible,
we observed that a large proportion of CD41+-Plt events were negative
for CD42 (73 ±7%, mean ±SD (n=5)). These could either be non-Plt
events such as MK-derived vesicles, non-viable Plts or Plts that have
lost expression of CD42. In order to increase the accuracy of the Plt
estimation, we investigated whether elimination of propidium iodide +
(PI) Plt events could reduce these undesired events. First, we confirmed
that fresh blood-derived Plts like viable cells exclude PI (<1% PI +).
Next, the analysis in 5 independent experiments demonstrated that
removal of PI+ Plt-events led to an increase in the proportion of CD41+Plt events co-expressing CD42, from 27 ± 7 to 37 ± 9% (P<.0001).
Importantly, this translated into a 50% reduction in false Plts counts
(CD41+CD42-), though it also led to a 10% reduction in CD41+CD42+Plts. Finally, we investigated whether the addition of a metalloproteinase
inhibitor (GM6001) would further increase the proportion of
CD42+CD41+-Plts, as reported for Plt concentrates. We found a dosedependant increase of CD41+CD42+ Plt (62 ±12% CD41+CD42+ at 20mM
(n=3)) with no significant toxicity, which provided a 1.3 ± 0.4 -fold
increase in Plt production compared to control culture (n=3). In summary,
the addition of a PI-negative selection gate improved the accuracy of
Plt analysis by FACS, whereas the addition of GM6001 to our MK-CB
cultures improved the Plt quality and Plt yield.
206/P60
CELL CYCLE ANALYSIS IN LIVE CELLS USING NOVEL
VYBRANT&REG DYECYCLESTAINS WITH VIOLET,
BLUE, AND GREEN EXCITATION
Jolene A. Bradford1, Pam Whitney2, Timothy Huang3,
Patrick Pinson3, Ching-Ying Cheng3, Stephen Yue3, William
L. Godfrey1
1
Molecular Probes, Inc., Flow Cytometry, Eugene, Oregon;
Invitrogen, Flow Cytometry, Madison, Wisconsin; 3Molecular
Probes, Inc, Chemistry, Eugene, Oregon
2
Cell cycle describes the progression of a cell through a cycle of division,
a process resulting in cell growth and separation into two daughter cells.
Flow cytometry testing is useful in describing the distribution of a
population of cells into the different nuclear phases of the cell cycle.
Analysis of the cell cycle is widely used in the study of cell growth and
defects in cell cycle regulation, oncology research, and DNA ploidy
determinations. These applications require dyes that bind to DNA in a
stoichiometric manner. With the exceptions of UV-excited dyes like
Hoechst 33342, cells have generally required fixation and
permeabilization as well as treatment with RNAse to obtain DNA-specific
cell cycle information.
The Vybrant&reg DyeCycle™ stains are
DNA-selective, cell membrane-permeant dyes that show greatlyenhanced fluorescence when bound to DNA and which can be excited
by 405 nm, 488 nm, or 532 nm lasers, depending on the dye. These
dyes show similar performance to Hoechst 33342 and DRAQ5: G0/G1
peak CV generally less than 6% and G2/G1 ratio greater than 1.8. The
dyes can be used on cells in the presence of media components, including
serum and divalent cations. Cell cycle analysis using the Vybrant
DyeCycle stains has been done on a wide variety of cells, including
Jurkat, CHO, 3T3, HL60, and peripheral blood lymphocytes, monocytes,
and neutrophils. Optimization of staining was performed for cell type,
cell concentration, dye concentration, staining time and temperature.
Dye staining has been combined with viability dyes to exclude dead
cells from analysis and has been used with antibody staining against
surface antigens. While the Vybrant DyeCycle stains cause some
retardation of cell division, they do not demonstrate the toxicity observed
in DRAQ5, and have been used to sort viable populations of cells from
G0/G1 and G2/M populations. Resolution of cell cycle information in
viable cells allows evaluation against the dynamic background of live
cell activity, as well as the ability to sort cells based on position in the
cell cycle.
207/P61
EFFECT OF X-IRRADIATION ON EARLY GASTRULAS
IN NORMAL MICE, DNA-REPAIR DEFICIENT MICE
AND MICE DEFICIENT IN CELL CYCLE CONTROL
Sarah Baatout1, Jasmine Buset1, Mieke Neefs1, Arlette
Michaux1, Bernard Chatelain2, Hanane Derradji1, Paul
Jacquet1
1
Belgian Nuclear Research Centre, Radiation Protection, Mol, ,
Belgium; 2Haematology UCL, Yvoir, , Belgium
Gastrulation in mice is known to be associated with a period of extreme
proliferation and differentiation. According to Heyer et al. (Genes &
Development, 14, 2072-2084, 2000), the potential cost to the embryo
of a very rapid proliferation rate is a high production of damaged cells.
The present research was performed in order to compare the radiationsensitivity of gastrulas in normal mice (C57BL and BALB/c), DNArepair deficient mice (scid and PARP-1 ko) and mice deficient in cell
cycle control (p53+/-). Pregnant females of the different strains were Xirradiated with 2.5 Gy of X-rays on day 8 of gestation. A number of
parameters were investigated 6 and 24 hours after irradiation, e.g.: total
area and length of the embryonic part of the gastrulas, cell and nuclear
morphology, cell area, cell cycle and caspase 3 activity. 5 hours after
X-irradiation, no statistical decrease in the total area and length of the
embryo was observed in scid, PARP and p53 +/- C57BL and BALB/c
mutants, except for scid gastrulas. However, 24 hours after X-irradiation,
total embryo area and length were significantly smaller compared to
control gastrulas, in all mutants as well as in the C57BL and the BALB/
c mice (though at a lesser extent in the two last strains). Morphological
studies performed on individual cells after pancreatin treatment and
following May-Grünwald Giemsa staining showed changes in the
general morphology of the cells after X-irradiation. Indeed, 5 hours
after the X-irradiation, the presence of blebs was noticed and cells with
condensed chromatin at the nuclear periphery appeared. 24 hours after
X-irradiation, bigger blebs, chromatin condensation, breaking up of
the nucleus and production of apoptotic bodies were observed. The
number of normal versus apoptotic and necrotic cells was quantified
and showed a significant increase of apoptotic cells 24 hours after Xirradiation in all the mutants. The mean cell size increased 5 hours after
X-irradiation (probably due to the presence of blebs) and decreased 24
hours after X-irradiation (due to the increased presence of pycnotic
cells and apoptotic bodies). We conclude that X-irradiation induced
cell death in the mouse gastrulas but the extent of radiation-induced
apoptosis depended on the type of mutation: the scid mutants were
shown to be the most radiation sensitive, followed by the PARP and the
p53 mutants. Further characterization enabling us to distinguish between
apoptosis and necrosis is currently being performed (in particular, the
annexin V-PI test and the TUNEL test).
This work is supported by a
research contract from the European Union (FIS5-2002-00029).
147
208/P62
PHENOTYPE AND FUNCTION OF CD56BRIGHT NK
CELLS GENERATED IN VITRO FROM HEMATOPOIETIC
PROGENITOR CELLS: IDENTIFICATION OF AN CD56+/
GRANZYME B-/PERFORIN- FUNCTIONALLY
IMMATURE HUMAN NK CELL SUBSET
Loris Zamai1, Claudia Masoni1, Laura Galeotti1, Barbara
Canonico2, Massimo Della Felice2, Fulvio Palma3, Stefano
Papa2
1
University of Urbino, Istituto di Istologia e Analisi di Laboratorio,
Urbino, Pesaro, Italy; 2University of Urbino, Cytometry and
Cytomorphology Center, Urbino, Italy; 3University of Urbino,
Istituto di Psicologia, Urbino, Italy
We have previously shown that CD34+ hematopoietic progenitor cells
cultured with SCF plus IL-2 or IL-15 differentiate into cytotoxic NK
cells. In the present report, we have phenotypically and functionally
characterized the CD56+ NK cells generated in vitro. After 20-30 days
of culture in the presence of Flt3L (or SCF) plus IL-15 (or IL-2) CD34+
hematopoietic progenitors differentiated into CD56bright NK cells
expressing CD161, CD56, CD117, CD122, CD132, IL-12Rbeta1, surface
activatory molecules: NCRs, 2B4 and NKG2D, TNF receptor members:
CD120a and b, CD95, TRAIL-R4 (a subset), TNF ligand members:
TRAIL (expressed by all CD56+ cells), CD95L (very low expression)
and TNF-alfa (a subset). Interestingly, only a subset of these cells
expressed CD11a, CD18, CD7, CD2, CD94/NKG2A and intracellular
cytotoxic molecules: granzyme B and perforin. CD56+ cells were then
purified and plated for further 15 days in secondary cultures with IL-15
or IL-2 with or without IL-21. After 15 days of culture, NK cells acquired
low levels of CD16, CD8, KIRs, and TRAIL-R2 (becoming sensitive to
TRAIL induced apoptosis), moreover, expression of LFA-1, CD94/
NKG2A and intracellular cytotoxic proteins increased during time,
indicating that IL-15 and IL-2 induced a phenotypic and functional
maturation. Altogether, the data indicate the sequence of surface and
intracellular NK cell markers acquired during differentiation of
CD56bright NK cells. Moreover, based on lytic protein expression we
have identified for the first time an immature CD56bright/Granzyme B/perforin- NK stage, that, nevertheless, expressed a membrane bound
form of TRAIL. This result suggest that during differentiation, NK cell
cytotoxic activity mediated by TRAIL is acquired earlier than that
dependent on granule release.
209/P63
USE OF FLOW CYTOMETRY IN THE VALIDATION OF
HUMAN MULTIPOTENT ADULT PROGENITOR CELLS
Amy C. Raber1, Mark Frey2, Pina Patel2, Emily Rebro2,
Robert Perry2, Robert Deans2, Wouter Vant Hof2
1
Athersys Inc., Cleveland, Ohio; 2Athersys Inc., Regnerative
Medicine, Cleveland, Ohio
MultiStemTM are non-embryonic stem cells capable of differentiating
in vitro and in vivo into mesodermal, endodermal, and ectodermal cell
lineages (including neuronal lineages). (Jiang Y et al, Nature, 2002,
418:41-49). In preclinical disease models, these cells have demonstrated
the potential for substantial therapeutic benefit in a variety of human
diseases. We have established protocols for large-scale expansion of
human bone marrow-derived MultiStemTM cultures with a stable
phenotype to levels allowing for sufficient clinical doses to enable clinical
entry. We routinely utilize flow cytometry as an important means for
standardized identification as well as for assessment of the differentiation
potential of the cells. We currently utilize panels of surface markers to
discriminate MultiStemTM from other non-embryonic cells or stem
cells. In addition, the cells are distinguishable from other adherent
bone marrow cultures, such as mesenchymal stem cells, by their
pluripotency, i.e., their ability to differentiate into cell lineages
representative of the three germ layers. We have established in vitro
differentiation assays that use statistically valid pass/fail criteria for
pluripotency using quantitative PCR. However, although qPCR provides
sensitive read-outs for the expression of lineage specific marker genes,
it does not describe other functional aspects of the differentiated cells
and does not quantify the number of cells in a culture undergoing
differentiation. For this purpose, we have combined functional adipocyte
differentiation assays with flow cytometry to measure fluorescent staining
of lipid vesicles as a quantitative assessment of adipogenic differentiation
on a cell-by-cell basis. This approach is being tested as a paradigm for
development of additional sensitive fluorescent-based cell-autonomous
148
assays with application in measurement
ectodermal and endodermal lineages as
phenotype and pluripotency assessment
development of standard processes for
and consistent cell product.
of cell differentiation into the
well. This standardization of
serves important needs in the
the manufacture of a reliable
210/P64
LIMB DEFECTS INDUCED BY X-IRRADIATION IN
MOUSE FOETUSES
Hanane Derradji1, Sofie Bekaert2, Rafi Benotmane1, Patrick
Van Oostveldt2, Sarah Baatout1
1
Belgian Nuclear Research Centre, Radiation Protection, Mol, ,
Belgium; 2Ghent University, Laboratory for Biochemistry and
Molecular Cytology, Gent, Belgium
In utero irradiation of the foetus during the period of organogenesis
can induce an increase in malformation. However, the mechanisms
underlying irradiation-induced teratogenesis are far from being
elucidated. In the present study, we focused on malformations of the
limbs to explore at the molecular level the complex development of
limb bud, following irradiation exposure. C57BL mice were exposed to
3 Gy X-rays in utero on day 12 of gestation during the period of
organogenesis of limb buds.
External examination under a
stereomicroscope of 19 day X-irradiated embryos revealed 100 % of
forelimb defects. Coexistence of different limb malformation variants
in a single limb segment was evidenced during the limb defect
characterization and classification steps. All foetuses were hypodactyl
(fewer than five digits on a limb) with various extent in the severity:
43% of the examined fetuses had 3 digits, 23 % exhibited 1 digit, 18,5
% 4 digits, 8 % 2 digits and 5,7 % had stump. Syndactyly (having two
or more fused digits) was highly present as 71 % of the foetuses showed
cutaneous syndactyly upon external examination while 99 % of them
revealed the presence of osseous syndactyly after the whole-mount
skeletal examination. Ectrodactyly (missing digit and metacarpal) and
aphlangy (missing digit but metacarpal present) concerned respectively,
80 and 49 % of the total foetuses observed. Further experiments are
currently under progress using microarrays to investigate early and late
gene expression modulations in limb buds from 12 day old embryos
irradiated with doses of irradiation up to 3 Gy. References 1- Derradji
H, Bekaert S, Van Oostveldt P, Baatout S. Comparison of different
protocols for telomere length estimation by combination of quantitative
fluorescence in situ hybridization (Q-FISH) and flow cytometry in
human cancer cell lines. Anticancer Res. 2005; 25(2A):1039-50. 2Derradji H, Baatout S. Apoptosis: a mechanism of cell suicide. In Vivo,
17(2): 185-92, 2003. 3- Bekaert S, Derradji H, Meyer TD, Michaux A,
Buset J, Neefs M, Mergeay M, Jacquet P, Van Oostveldt P, Baatout S.
Telomere shortening is associated with malformation in p53-deficient
mice after irradiation during specific stages of development. DNA Repair.
2005; 4(9):1028-37. 4- Bekaert S, Derradji H, Baatout S. Telomere
biology in mammalian germ cells and during development. Dev. Biol.,
274, 15-30, 2004. 5- Baatout S, Derradji H. Cytometric methods to
analyze radiation effects. J. Biol. Regul. Homeost. Ag., 18(2):101-5,
2004.
211/P65
COMPARISON OF CFSE AND PKH26 WITH CELLVUETM
CLARET, A NEW 675NM-EMITTING PROLIFERATION
DYE
Andrew Bantly1, Brian D. Gray2, Elizabeth Breslin2,
Katharine A. Muirhead3, Betsy M Ohlsson-Wilhelm4, Jonni
Moore1
1
University of Pennsylvania, Pathology and Laboratory Medicine,
School of Medicine, Philadelphia, Pennsylvania; 2PTI Research,
Inc., Exton, Pennsylvania; 3SciGro, Inc., Madison, Wisconsin;
4
SciGro, Inc., Cambridge, Massachusetts
GOAL: Cell tracking dyes such as PKH26 and CFSE have proven useful
in numerous applications including assessment of cell proliferation. We
sought to determine whether CellVueTM Claret (formerly PTIR289), a
far-red fluorescent membrane intercalating dye, could be used as an
alternative to PKH26 and CFSE in multicolor proliferation studies on a
standard 2 laser/4 color BD FACSCalibur. METHODS: Using peripheral
blood mononuclear cells (PBMCs) from 4 different donors, we compared
PTIR289 with PKH26 and CFSE in terms of: 1) ability to monitor
proliferation in CD3+ lymphocyte subsets and 2) compatibility with 7-
AAD as a viability marker. The proliferative response of PTIR289-,
PKH26- or CFSE-stained PBMCs to stimulation with soluble antiCD3+IL-2 was evaluated at 0, 24, 72 and 96 hours. RESULTS: In high
responding individuals, post-stimulation CD3 + T cell viabilities and
proliferative responses were comparable for all 3 dyes, using either
proliferative fraction or precursor frequency as the measure of
response.More variation was observed in low responders, at least in part
due to differences in response kinetics, but all 3 dyes detected similar
patterns of response. CONCLUSION: These preliminary studies indicate
that CellVue TM Claret can be a useful alternative to PKH26 and CFSE
for cell tracking and proliferation monitoring studies on a 2-laser BD
FACSCalibur, providing flexibility in choosing probes for other
functional or phenotypic evaluations and overcoming compensation
difficulties often found with PKH26 and CFSE.
212/P66
ISOLATION OF ENRICHED PROGENITOR CELLS IN
THE PLANARIAN SCHMIDTEA MEDITERRANEA BY
FLOW CYTOMETRY
Namson-Hawk Oh1, James C. Jenkin2, Wayne F. Green3,
Alejandro Sánchez Alvarado2
and fluorescence microscopy to analyse the effect of a Src kinase
inhibitor, SU6656, on the polyploidization of the human K562 and M07e Mk cell lines. Examination of K562 cell nuclear material stained
with DAPI revealed the presence of distinct, well polylobulated nuclei.
Based on tubulin expression, these cells progressed normally from
prophase to anaphase but omitted cytokinesis. As to M-07e treated cells,
similar analyses revealed the accumulation of more compacted
polylobulated nuclei. Consistent with the apparent absence of mitotic
spindles, abnormal microtubule formation was observed in these cells,
indicating that aberration in the mitotic process occurred as early as
during prometaphase, although anomalies in prophase are not excluded.
Since SU6656 promotes polyploidization in Mk cells, we next wanted
to examine the effect of SU6656 on non-Mk cell lines. Cell-cycle analysis
of SU6656-treated B-cell-derived lymphoma cells indeed revealed a
G1 block in Raji and Namalwa lines, whereas cells from another
lymphoma line, DB, accumulated in G2/M. Remarkably, longer
incubation in the presence of the kinase inhibitor resulted in the
accumulation of viable polyploid DB cells. Microscopic examination of
these cells revealed the presence of polylobulated nuclei similar to those
observed in K562 cells. Thus, in addition to its known utility for the
study of Mk differentiation, our data suggest that prolonged exposure
to SU6656 induces polyploidy in B-cell-derived lymphoma cells.
1
University of Utah, Core Laboratories, School of Medicine, Salt
Lake City, Utah; 2University of Utah, Neurobiology and Anatomy,
School of Medicine, Salt Lake City, Utah; 3University of Utah,
Medicine/Hematology, School of Medicine, Salt Lake City, Utah
The genetic control and physiologic function of planarian multipotent
progenitor cells are being investigated as a means of providing possible
insight into the regeneration of organs and tissues in multicellular
organisms as well as stem cell function. The planarian possesses a
population of totipotent stem cells, called Neoblasts, which are
responsible for the robust ability of planarians to regenerate. Neoblasts
have been shown to be the only mitotically active cells in S. mediterranea
and exposure to gamma irradiation has been shown to prevent
regeneration. We describe a modified Hoechst 33342 Side Population
assay, with which we have been able to show that radiation sensitive cell
populations can be detected and sorted for further study. Worms were
cut transversely, anterior and posterior to the pharynx, to expose the
digestive tract repeatedly and incubated calcium-free media to prepare
single cell preparations. Viability of the dissociated planarian cells, as
determined by propidium iodide exclusion, is consistently better than
90%. Staining with Hoechst alone or with Hoechst plus calcein identified
two cell populations, referred to as X1 and X2, which were clearly
susceptible to deletion by gamma irradiation. The X1 population can
be isolated using Hoechst alone while the X2 population is more readily
isolated with Hoechst and calcein. Sorted X1 and X2 populations
exhibited small size, large nucleii and minimal cytoplasm; characteristics
consistent with Neoblasts. Initial results indicate that the X1 cells appear
to have a higher DNA content than X2 cells and express PIWI-1, PIWI2 and Cyclin B while Cyclin B expression is absent in X2 cells. Further
characterization of the gene expression and function of the X1 and X2
populations and their role in regeneration is planned.
213/P67
SU6656 INDUCES POLYPLOIDIZATION IN
MEGAKARYOCYTIC AS WELL AS IN CERTAIN
LYMPHOMA CELL LINES
Carl Simard1, Nathalie Dussault2, Serge Côté1, Sonia Néron3
1
Héma-Québec, Ste-Foy, Quebec, Canada; 2Héma-Québec, R&D,
Cellular Engineering, Sainte-Foy, Quebec, Canada; 3Laval
University, Ingénierie Cellulaire, Héma-Québec, Sainte-Foy,
Quebec, Canada
Cells that fail to divide following DNA replication activate a p53dependent mechanism that arrests them in the next G1 phase and thereby
inhibits their propagation as polyploids, a cell context which is postulated
to generate genetic instability. However, certain mammalian cell types
can naturally develop polyploidy, such as the megakaryocyte (Mk), a
bone marrow cell that generates platelets, and where increase in ploidy
is part of its differentiation process. Although the underlying mechanism
controlling polyploidization remains largely unknown, certain chemicals,
such as phorbol esters, have been shown to be capable of initiating in
Mk cell lines a series of events closely similar to those that occur during
normal Mk development. In the present work, we have used cytometry
214/P68
HEMATOPOIETIC CELLS CULTURED UNDER MILD
HYPERTHERMIA UNDERGO AN ACCELERATED CELL
DIFFERENTIATION AND PROLIFERATION KINETICS
Jean Francois Boucher1, Nicolas Pineault1
1
Héma-Québec, R&D, Québec, Quebec, Canada
Our laboratory focuses on the development of culture conditions for
the production of platelets from cord blood (CB) CD34+-cells. Specific
to our strategy, is the culture of the CB cells at 39C, since we have
previously reported that this led to an increase in megakaryocyte (MK)
and platelet production. The present study had for objective to investigate
the impact on the cell cycle kinetics of culturing CB cells at 39C. In
brief, CB-CD34 + cells were cultured in serum-free media either at 37
(control) or 39C in two distinct cytokine cocktails known to promote
hematopoietic progenitors (A: SCF, FL, TPO, IL-6) and MK cells (B:SCF,
TPO, IL-9, IL-6) expansion. Phenotypic and cell cycle kinetics analysis
of the cultures were done by FACS using lineage-specific markers and
propidium-iodide staining, respectively. Expansion and differentiation
were followed on a daily basis for 7 and 14 days in cocktail A and B.
With the cytokine-rich cocktail A, mild hyperthermia led to an increase
cell expansion of total nucleated cell (TNC) (129,0 ± 1,4 at 37 VS 212,0
± 2,8 at 39), CD34+ cells (52,6 ± 0 at 37 VS 84,4 ± 4,4 at 39), CD41+
megakaryocytes (17,2 ± 1,8 at 37 VS 46,0 ± 8,1 at 39) and CD235a +
erythrocytes (2-fold). The impact on proliferation was rapid, as TNC
expansion was 25% higher at 39C at day 3 (n=2). The mean doubling
time for these cultures was 1.1 hour shorter at 39C. The impact on
CD41+ MK doubling time was even more pronounced with a 4.4 hours
reduction. As previously observed, MK differentiation kinetics were
greatly accelerated at 39C compare to 37 in cocktail B, with the proportion
of mature MK (CD41+CD42+) being 1.7 ±0.3-fold greater at 39C at day
7 (n=3, P<0.004), and a 2–fold increase in CD42+ MKs at day 7 (15.5
±2.5 at 37 VS 26.6 ±4.9 at 39 (mean ± SD (n=3)). To link the faster
doubling times with the boost in expansion and differentiation, we
analysed the cell cycle progression of the human erythroleukemia line
K562 to identify which steps of the cell cycle was most affected. Mild
hyperthermia had a modest effect on the expansion of K562 cells, with
exposition over 5 days being detrimental to viability. Thus, we analysed
the cell-cycle kinetic of K562 cells grown for up to 4 days at 39C. The
number of cells in G1 phase were significantly lower at 39C (52,88
±2,37 % at 37 VS 49,79 ±1,7% at 39) due to an increase of cells in the
S/G2/M fraction (45,34 ±2,97% at 37 VS 47,55 ±2,82% at 39 (n=3,
p<0,05)). We are presently assessing whether similar changes are taking
place in CB cells cultured at 39C. Altogether, our results suggest that the
overall increase in the cell kinetics (differentiation and expansion)
associated with culture at 39C, is due in part to a faster cell cycle
progression through the G1 phase and/or the G1/S check point.
149
215/P69
MATHEMATICAL MODEL OF IN VITRO
MEGAKARYOPOIESIS
Younes Leysi-Derilou1, Carl Duchesne2, Marie-Christine
Hains1, Nicolas Pineault3, Alain Garnier4
1
Laval University&Héma-Québec, Quebec city, Quebec, Canada;
Laval University, Chemical Engineering, Quebec city, Quebec,
Canada; 3Héma-Québec, Quebec, Quebec, Canada; 4Laval
University, CREFSIP, Quebec city, Quebec, Canada
2
Megakaryopoiesis is a complex process by which hematopoietic stem
cells (HSC) differenciate into megakaryocytes (MK), undergo
endomitosis, massively produce extensions called proplatelets and
ultimately yield blood platelets. Throughout this process the cells are
also proliferating, and it is not clear if MK expansion is mainly due to
HSC differentiation or MK proliferation as such. To answer this question,
we have developed a simple kinetic model for megakaryopoiesis which
takes into account the proliferation, differentiation, and death rates of
each precursor. While cell differentiation and death coefficients were
assumed constant, a logistic relation was used to account for the
proliferation kinetics. The model was fitted to FACS data obtained at
different time points of a HSC culture isolated from cord blood and put
in conditions favoring megakaryopoiesis. The simulation represented
well the experimental results (Figure 1). The values of the coefficients
obtained clearly show that MK proliferation contributes importantly to
the expansion of these cells.
216/P70
IMPROVED MULTIPARAMETER FLOW CYTOMETRIC
DNA CONTENT ANALYSIS IN NEUROBLASTOMA
USING DRAQ5
Katrien Swerts1, Yves Benoit1, GenevièVe Laureys1, Jan
Philippé2
marker. We first compared whole sample DRAQ5 and PI cell cycle
analyses using normal peripheral blood samples. DRAQ5 and PI showed
almost identical stoichiometric DNA binding characteristics. However,
DRAQ5 produced slightly larger coefficients of variation (around 3.0;
still within the acceptable range). Next, the DNA content of NB cell
subpopulations, spiked in peripheral blood, was analyzed by performing
simultaneous GD2/CD45 and DRAQ5 staining. Since light scatter and
antigen expression were completely preserved, DRAQ5-based DNA
content analysis is easily performed. The DNA content of as little as 100
NB cells per 2.105 normal hematopoietic cells could be determined in a
reliable way. By contrast, this was completely impossible using the
conventional PI-based assay. In conclusion, DRAQ5 allows easy and
rapid DNA content measurements of NB subpopulations that are clearly
defined by light scatter and immunophenotype.
217/P71
THE FREQUENCY OF MICRONUCLEI IN HUMANS
MEASURED BY FLOW: IMPACT OF DIET
Natalia Kotova1, Jan Grawé2
1
Stockholm University, Dept. Genetics, Microbiology and
Toxicology, Stockholm, Sweden; 2Uppsala University, Rudbeck
Laboratory, Uppsala, Sweden
Diet is one of the main routes of exposure to genotoxic agents. Therefore,
it may be possible to decrease the amount of genetic damage sustained
by an individual through the choice of diet. Thus it is desirable to
determine how diet impacts on the amount of induced damage. We have
used enumeration of micronuclei (MN) in transferrin-positive immature
peripheral blood reticulocytes by flow cytometry as a quantitative
measure of genetic damage. The technique is based on the fact that very
young micronuclei-containing reticulocytes (MN-Ret) in humans survive
approximately 20 minutes in the blood stream before been removed by
spleen. During this time the reticulocytes still express the transferrin
receptor (CD71). These cells can be enriched by the use of
immunomagnetic separation for this receptor. Staining with Hoechst
33342 (for DNA-content) and Thiazole Orange (for RNA-content)
enables rapid quantification of MN-Ret by flow cytometry. Peripheral
blood samples were investigated from 26 healthy donors in the age
range 20-30 years. Based on a questionnaire, the donors were divided
into two groups: non-vegetarians group included fish and/or red meat
eaters (group A, 14 donors) and vegetarians (group B, 12 donors). The
frequencies of MN-Ret varied between 0.42 and 1.49% (mean 0.96%)
in group A and 0.37 and 1.03% (mean 0.68%) in group B. The group
A showed significantly higher frequencies than the group B (p<0.03,
two-tailed Student´s t-test). Although we have indications for a correlation
between MN frequencies and diet, further studies are needed to establish
that the observed relationship is a direct result of ingested mutagens. A
major advantage of present method is that due to the high sensitivity it
can be used to detect small differences in the MN level, allowing the
study of effects of low exposures. Moreover, MN-containing
erythrocytes are rapidly eliminated from the blood stream making possible
the use of each individual as its own control by sampling before and
during, or after, a presumed exposure. Thus the present method allows
further studies where donors may be sampled over time while switching
between diets.
1
Ghent University Hospital, Department of Pediatric Hematology
and Oncology, Ghent, Belgium; 2Ghent University Hospital,
Department of Clinical Chemistry, Microbiology and Immunology,
Ghent, Belgium
The DNA content of neuroblastoma (NB) cells is a prognostic parameter
predicting response to treatment and outcome, especially in infants less
than 1 year of age with advanced stage disease. Flow cytometry, utilizing
intercalating dyes such as propidium iodide (PI), has been extensively
used for DNA content analysis. However, the majority of these analyses
were performed on whole tumor and bone marrow samples without
taking the effect of ‘contaminating´ hematopoietic cells into account.
Consequently, simultaneous analysis of cellular DNA content and
immunophenotype would greatly improve the reliability of ploidy
measurements by enabling the identification of NB cells in a complex
mixture. The broad emission spectrum of PI makes it difficult to use
more than one fluorochrome for simultaneous antigen binding.
Therefore, we developed a three-color flow cytometric assay using
DRAQ5, a novel synthetic anthraquinone dye, for cell cycle analysis.
NB cells are identified based on their GD2 disialoganglioside expression.
CD45, present on all cells of hematopoietic origin, is included as negative
150
218/P72
SPIRULINA AS FOOD SUPPLEMENT FOR LONG-TERM
MANNED SPACE MISSIONS : EFFECT ON CANCER
CELLS
Sarah Baatout1, Hanane Derradji1, Sofie Bekaert2
1
Belgian Nuclear Research Center, Radiation Protection, Mol,
Belgium; 2Gent University, Molecular Biotechnology, Gent,
Belgium
The cyanobacteria A. platensis (also named spirulina) is known for its
potential health effects and is reported in the literature to diminish
cholesterol level, to be active against diabetes, hypertension, anemia
and iron bioavailability, to reduce kidney toxicity from drugs and heavy
metals, to help to loose weight, to inhibit HIV replication and to reduce
the effects induced after Chernobyl radiation. Some of the beneficial
effects are attributed to its natural amount of antioxidants (spirulina is
the richest beta-carotene (main source of vitamin A) food known (10x
> carrots) as well as iron (58 x > raw spinach and 28 x > raw beef liver).
However, spirulina also contains toxins that could be cytotoxic for
humans. Spirulina is also of potential interest in the context of longterm manned missions in the European Space Agency (ESA) and
National Aeronautics and Space Administration (NASA) research
programmes. In this study, we were interested in studying the threshold
of intrinsic cytotoxic effect of spirulina on human cancer cells and its
cell type dependency. For that purpose, we used flow cytometry to
estimate apoptosis and necrosis in three human leukaemic cell lines
(HELA : cervix carcinoma; IM-9 : multiple myeloma; K562 : chronic
myelogenous leukaemia). Cells were cultured in the presence of water
extract of spirulina (concentrations ranging from 0 to 500 µg/ml) between
15 to 40 hours. Apoptosis and necrosis were evaluated by the annexinV-PI staining, cell size and granularity. Early apoptosis was checked by
analysing the maintenance of mitochondrial membrane potential
(DioC6(3)) and the production of superoxides (hydoethidine) whereas
advanced apoptosis was studied by measuring the propensity of the
sub-G1 peak, esterase activity (fluorescein diacetate) and intracellular
pH (carboxyfluorescein diacetate). Intracallular calcium concentration,
intracellular thiols and H2O2 concentrations were also monitored. The
three cell lines had a different sensitivity to spirulina extracts (HELA
most resistant > K562 > IM-9 most sensitive). These results suggest
that, depending on the concentration, spirulina extracts could enhance
apoptosis in cancerous cell lines. This may open a perspective for cancer
therapy at the condition to also address the intrinsic toxicity of spirulina
on normal cells. This work is partially supported by ESA/ESTEC
(contract number n°15680/01/NL/ND).
219/P73
SPACE MICROBIOLOGY: PHYSIOLOGICAL BEHAVIOUR
OF BACTERIA
Sarah Baatout1, Larissa Hendrickx1, Natalie Leys1, Max
Mergeay1
1
Belgian Nuclear Research Center, Radiation protection, Mol,
Belgium
The detection and monitoring of bacteria that are present in space crafts
is crucial for biosafety of prolonged manned space missions. Microbial
activity in closed space environments (e.g. space vehicles, space stations,
planetary bases) can negatively affect crew health (e.g. infections) or
material structure (e.g. biodegradation, biocorrosion). Nonetheless,
bacteria can also be essential for food production and waste recycling
(e.g. air revitalization, water purification, waste degradation) in view of
long-term manned space missions (e.g. the MELiSSA project for MicroEcological Life Support System Alternative). The organisms inhabiting
the MELISSA system need to perform their tasks as optimally as possible.
A number of stresses such as temperature variation, oxidative and pH
stress can affect the capabilities of the microorganisms as well as the
efficiency of the whole system. Furthermore, two MESSAGE experiments
(Microbial Experiments in the Space Station for the Analysis of Gene
Expression) were performed during 2 separate visits to the International
Space Station (ISS). In this presentation, we will review the effect of
temperature stress (temperatures from -170°C to 70°C), oxidative stress
(H2O2 concentrations from 0 to 880 mM), acid and alkaline stress
(ranging from pH 2 to 12) and/or ‘space flight´ stress (10 day-stay in
the International Space Station ) on three types of bacteria : Cupriavidus
metallidurans, Rhodospirillum rubrum and Arthrospira species. Cell
membrane permeability and potential, intracellular esterase activity,
intracellular reactive oxygen species concentration and intracellular pH
were assessed by flow cytometry to evaluate the physiological state and
the overall fitness of individual bacterial cells under the different stress
conditions. We report that the bacterial strains exhibited varying
responses in function of the type of stress applied and the type of
bacteria. A moderate physiological change was observed at temperatures
below or higher than the optimal culture temperature, at H2O2
concentrations above 13.25 mM, at two units pH under the optimal
culture pH or after a spaceflight. Membrane permeability and potential,
esterase activity, intracellular pH and superoxide anion production were
significantly modified at high or low temperatures, low pH and high
H2O2 concentrations. Our experiments show that a range of significant
bacterial physiological alterations occurs under stress conditions and
that fluorescent staining methods coupled with flow cytometry are useful
for monitoring subtle changes observed after various stresses including
a spaceflight. This work is supported by ESA/ESTEC, ESA/PRODEX
and the Belgian Federal Office for Sciences, Technology and Culture.
220/P74
SHORT-TERM VARIABILITY OF ULTRAPLANKTON IN
THE NORTHWESTERN MEDITERRANEAN IN LATE
SUMMER 2004. EVIDENCE FOR PULSED
MINERALISATION IN THE WATER COLUMN
Michel Denis1, Melilotus Thyssen2, Gérald Grégori3, Cindy
Tavernier4
1
Centre d’Océanologie de Marseille, Lab de Microbiologie,
Géochimie et Ecologie Marines, Marseille Cedex 9, France; 2Centre
National de la Recherche Scientifique, Laboratoire de Microbiologie,
Géochimie et Ecologie Marine, Marseille, France; 3Université de la
Méditerranée, Marseille, France; 4Université de la Méditerranée,
Laboratoire de Microbiologie, Géochimie et Ecologie Marines,
Marseille cedex9, France
The relationship between pelagic processes and exported carbon flux is
highly complex. Interactions between physical and biological controls
may occur on top of possible bathymetry forcing and so at different
temporal and spatial time scales. In particular, the role of the diverse
heterotrophic organisms in carbon mineralisation raises questions that
remain to be clarified. Studies aiming at carbon export prediction usually
are not considering the diversity of heterotrophs, their community
structure, the succession of dominant species and their function. This is
a major issue to understanding the impact of heterotrophic processes on
matter and energy fluxes. In a previous study (Denis et al., 2003), an
hypothesis was elaborated suggesting the possibility of pulsed
mineralisation triggered by diel vertical migration of zooplanktonic
organisms. In the frame of a national program (PECHE) investigating
the production and exportation of carbon and more specifically the
control by heterotrophs at short-time scales, we studied by flow cytometry
the short-term variability of ultraplankton abundance and tested the
sustained wave train hypothesis in the 0-1200 m water column. This
study took place during the DYNAPROC 2 cruise (12 September - 17
October 2004) in the northwestern Mediterranean, away from the
Ligurian current to minimise advection. Several time series were
conducted in the upper 200 m with a frequency of 6 h during 5 days
and 3 h over 36 h and also down to 1200 m with a frequency of 3 h
during 36 h. Results will be presented to illustrate the short-term
variability of ultraplankton in the upper layer and the heterogeneity of
the bacterial community and its structural changes with depth monitored
by staining their nucleic acids. The bacterial viability was also investigated
for each sample by using the nucleic acid double staining (NADS) of
Grégori et al. (2001). Data from the time series support the existence of
pulsed mineralisation in the water column that would be triggered by
diel vertical migration, shedding a new light on heterotrophic activity
in the aphotic layer. References Denis M., V. Martin, A. Momzikoff, G.
Gondry, L. Stemmann, S. Demers, G. Gorsky, V. Andersen. (2003)
Pulsed remineralisation in the northwestern Mediterranean Sea: an
hypothesis. J. Mar. Syst. 39: 19-41. Grégori G., Citterio S., Ghiani A.,
Labra M., Sgorbati S., Brown S. and Denis M. (2001) Resolution of
viable and membrane-compromised bacteria in freshwater and marine
waters based on analytical flow cytometry and nucleic acid double
staining. Appl. Environ. Microbiol. 67: 4662-4670.
221/P75
FIRST FLOW-CYTOMETRIC DETERMINATION OF
PICOPHYTOPLANKTON ABUNDANCE IN THE HUDSON
BAY AND ADJACENT WATERS: UNEXPECTED
DOMINANCE BY PICOCYANOBACTERIA
Claude Belzile1, Michel Gosselin1, Joannie Ferland1,
MéLanie Simard1
1
Institut des sciences de la mer de Rimouski, Rimouski, Quebec,
Canada
Picocyanobacteria are the most abundant autotrophs in the World oceans.
Marine phycoerythrin-rich cyanobacteria with diameter less than 2 ìm
are generally identified as Synechococcus . Flow-cytometric
determinations of picophytoplankton abundance in the north polar waters
are very few, with only three papers presenting data for sites north of
60 oN. Here we present the first flow-cytometric determination of picoand nanophytoplankton abundance in the Hudson Bay and adjacent
waters during fall at latitude ranging 55-64 o N. In agreement with the
general view that picocyanobacteria are nearly absent from the Arctic
Ocean, these prokaryotic cells were found in low numbers in the waters
of Arctic origin entering the Foxe Basin and Hudson Strait (the points of
151
entry of marine waters into the Hudson Bay). Surprisingly,
picocyanobacteria were numerically dominant in the Hudson Bay, and
reached concentrations as high as 35000 cells per mL in the western
part of the Bay. Typically, such high concentration of picocyanobacteria
in the Ocean is found in nutrient-rich coastal or mesotrophic waters.
The highest concentration of picocyanobacteria occurred at mid-range
salinities (salinity 28-30), and picocyanobacteria were less abundant in
regions strongly influenced by river discharge (near the plume of the
Great Whale and Nelson rivers, and in James Bay). Flow-cytometry
have revealed a new and unexpected oceanographic region of
picocyanobacteria dominance in a cold water environment.
223/P77
IMMUNOPHENOTYPING NEOPLASTIC HAEMOCYTES
OF MYA ARENARIA : A NEW APPROACH IN
DIAGNOSTICS OF MOLLUSC DISEASES
Maryse M. Delaporte1, Patty McKenna1, Franck Berthe1
1
University of Prince Edward Island, Department of pathology and
microbiology, Charlottetown, Prince Edward Island, Canada
Haemic neoplasia of the soft shell clam, Mya arenaria, is characterized
by the proliferation of abnormal neoplastic haemocytes in both the
circulating and tissular compartments. Neoplastic cells typically display
a relatively enlarged nucleus, high nucleus-to-cytoplasm ratio, and
increased frequency of mitotic figures. Detection is traditionally
performed by hemato-cytology and/or histopathology. Flow cytometry
protocols have been mainly developed to diagnose the disorder based
on haemocyte DNA content. Although, monoclonal antibodies had been
raised against the normal and transformed haemocytes of clams, no
protocol was developed to establish the correlation between DNA content
and antigen expression in the course of the disease. Flow cytometry
enabling concomitant use of different fluorescent dyes, the aim of our
study is to develop an immunophenotyping protocol for disseminated
neoplasia diagnosis in the soft shell clam Mya arenaria coupling
propidium iodide for DNA quantification and the Mab1E10 raised
against the neoplastic cells. Preliminary results will be presented at the
meeting.
224/P78
THE FLOW CYTOMETRY OF BACILLUS CEREUS
ENDOSPORES: CHARACTERISING AND QUANTIFYING
DAMAGE AND GERMINATION RATE
Ultan Philip Cronin1, Martin Gerard Wilkinson1
1
Ireland
The food-poisoning microorganism, Bacillus cereus , encountered in
rice-based meals (Little et al., 2002), forms resistant endospores, which
can survive many food preservation techniques and subsequently
germinate under appropriate conditions (Coroller et al., 2001). A rapid
method which allows the quantification of the degree and nature of
damage suffered by endospores under various preservation techniques
and which also quantifies the rate of germination would be of value in
developing food safety strategies to counteract this bacterium. The
development of a flow cytometry (FCM)-based method for these
applications is described. Quantifying the degree and nature of damage
involved treatment of endospores with six compounds (D-alanine, EDTA,
ethanol, gluteraldehyde, hydrogen peroxide and iodine) which arrest
germination at defined stages, three treatments which remove either
exosporium, protein coat or cortex and a demineralization treatment.
Endospores were then stained with SYTO 9 and propidium iodide (PI)
and analyzed by FCM. The rate of endospore germination and
quantification of the numbers of endospores with esterase activity
associated with this event involved staining with carboxyfluorescein
diacetate (CFDA) and Hoechst 33342. Endospores subjected to the
various treatments displayed discrete patterns of SYTO 9 and PI
permeability and, in some cases, altered side scatter (SSC) profiles. The
FCM method developed for assessing the rate of endospore germination
compared favourably with the standard microbiological nutrient agar
plate count method ( r = 0.706, p < 0.05). Data from a heat stress
survival curve generated from conventional plating techniques was
compared with data generated by using either a gate on high green (CF)
fluorescence events or a gate on high violet (Hoechst 33342)
fluorescence. A strong correlation existed between the standard
microbiological method and green fluorescence ( r = 0.892, p < 0.01).
152
However, the relationship between survival and high permeability to
Hoechst 33342 was more complex and could be described by a binomial
equation (R 2 = 0.8094). The methods developed allow rapid direct
estimation of numbers of intact B. cereus endospores and those
possessing the ability to germinate. Permeability profiles may be applied
to assess the extent and nature of damage caused to endospores during
cooking, pasteurisation, or exposure to food preservatives. Coroller,
L., Leguerinel, I., and Mafart, P. (2001). Applied and Environmental
Microbiology 67(1), 317-322. Little, C. L., Barnes, J., and Mitchell, R.
T. (2002). Communicable Disease and Public Health 5(4), 289-298.
225/P79
INTEGRATION OF FLOW CYTOMETRY WITH SINGLE
Laura A. Adang1, Chris Parsons2, Dean H. Kedes3
1
University of Virginia, College and Graduate School of Arts and
Sciences, Charlottesville, Virginia; 2UVA, Internal Medicine,
Charlottesville, Virginia; 3University of Virginia, Myles H. Thaler
Center for Aids and Retrovirus Research, Charlottesville, Virginia
Kaposi´s sarcoma-associated herpesvirus (KSHV) is the etiologic agent
of Kaposi´s sarcoma (KS), the most prevalent neoplasm in acquired
immune deficiency syndrome (AIDS) patients, as well as two other
lymphoproliferative disorders, primary effusion lymphoma and
multicentric Castleman´s disease. Predominantly, KSHV persists within
host cells in a semi-quiescent state called latency. Maintenance of latency
is dependent on expression of a virally encoded protein, latencyassociated nuclear antigen (LANA). Immunofluorescence assays (IFA)
following de novo KSHV infection in culture as well as in situ IFA of
KS tumors have shown that LANA localizes to discrete nuclear foci in a
pattern that is diagnostic of KSHV-infected cells. To more precisely
measure KSHV infection, we have utilized a new technology (Image
Stream 100, Amnis) that combines the quantitative and high throughput
benefits of flow cytometry with the visual analysis of traditional
immunofluorescence microscopy. The advantage of this approach for
KSHV infection is that it allows for visualization and automated gating
of cells displaying the distinctive and, therefore, specific staining pattern
of LANA within a cell population. Our results suggest that both
fluorescence intensity and nuclear spot visualization following reaction
with a fluorochrome-conjugated monoclonal antibody to LANA correlate
directly with viral episome copy number. Furthermore, this approach is
effective in detecting even low levels of KSHV infection in a variety of
primary and cultured cells. Use of additional fluorescent antibodies to
cell surface markers and other viral proteins allows for characterization
of individual cells with differing levels of infection or viral “burden”.
Although requiring further study, this technique may prove to be a
powerful tool for the analysis of the effects of whole virus on cell
systems and tissues and has potential for clinical screening for KSHV
and other viral infections.
226/P80
APPLICATION OF FLOW CYTOMETRY TO
INVESTIGATE THE EFFECTS OF CELL ATTENUATION
METHODS ON PERMEABILITY AND INTRACELLULAR
ENZYME RELEASE FROM LACTOCOCCUS LACTIS
SUBSP. LACTIS 303
Imelda A. Doolan1, Martin GERARD Wilkinson2
1
University of Limerick, Life Sciences, Limerick, Ireland;
Ireland
2
Lactococcus lactis subsp. lactis 303 (303) is an important commercial
Cheddar cheese starter culture in widespread use in Ireland. This strain
has been selected for it’s consistent acidification profile and resistance
to bacteriophage but generally requires to be combined with 2-3 other
strains during cheesemaking. When 303 is used as a single strain starter,
the resulting cheese is bitter due to its high viability, low level of autolysis
and poor release of intracellular ripening enzymes, especially peptidases.
Hence, identification of methods to enhance intracellular enzyme release
from this strain may have applications for enhancement of flavour and
overall acceleration of cheese ripening. In this study, three chemical
permeabilization agents were evaluated for their impact on cell viability,
autolysis and intracellular enzyme release. Cell permeabilisation was
induced by treating cells with; 50%, 70% and 100% isopropyl alcohol
(IPA) for 30 minutes, 1 mmol L-1, 2 mmol L-1 and 3 mmol L-1 CTAB for
15 minutes and 0.1%, 0.2% and 0.3% SDS for 15 minutes. 303 cells
were grown in either L-M17 broth (Oxoid) or in 10% Reconstituted
Skim Milk (RSM, Oxoid) to stationary phase and harvested by
centrifugation. Subsequently, cell pellets were re-suspended in chemical
permeabilizing agents, stained using using a Live/Dead Baclight kit and
analyzed using Flow Cytometry. Autolysis and release of the intracellular
peptidases as a result of the various attenuation methods was monitored
by measurement of Pep X and Pep N activity in cell pellets recovered
after permeabilization treatments. Flow cytometry (FCM) differentiated
three discrete sub-populations corresponding to; R1 (intact, untreated)
R2, permeabilised (CTAB, SDS-treated) or R3 dead (IPA-treated) cells.
Regardless of the source or concentration of permeabilizing agent, these
three sub-populations were present for cells grown in either L-M17 or
10% RSM. Treatment of cells generally resulted in ~90% of the
population transiting from R1 to R2/R3 regions. Levels of intracellular
enzymes released from cells indicated that all permeabilizing treatments
resulted in an increase in autolysis or enzyme acessibility indicated by
elevated Pep X activity compared with control cells. In particular, CTAB
and SDS treatments had a significant positive effects on the release of
Pep X activity with fifteen fold increase in Pep X activity compared
with untreated cells. However, increasing concentrations of CTAB or
SDS did not result in release of higher levels of Pep X perhaps due to
enzyme denaturation. This study indicated the potential for FCM in
conjunction with intracellular enzyme assays to design a permeabilization
method to increase enzyme release from a non-autolytic cheese starter
strain.
227/P81
PHYSIOLOGICAL STUDIES OF THE COPPER
RESISTANCE OF CUPRIAVIDUS METALLIDURANS BY
FLOW CYTOMETRY
Sébastien Van Aelst1, Abdelmalek Bahri1, Max Mergeay2,
Françoise Van Vliet3, Sarah Baatout2
1
Université Libre de Bruxelles, Laboratory for Microbiology,
Bruxelles, Belgium; 2Belgian Nuclear Research Centre, Radiation
Protection, Mol, Belgium; 3Institut de Recherches Microbiologiques
Jean-Marie Wiame, Bruxelles, Belgium
Flow cytometry is a well established technology to study the physiology
of Eukaryotic cells by the use of an arsenal of different dyes. Since
recently, flow cytometric methods have also been adapted to
microbiology. In this context, our laboratory is currently studying
physiological changes induced by various stresses in bacteria using
flow cytometric methods and has recently shown major changes in
Cupriavidus metallidurans CH34 when submitted to a thermal stress
(3). C. metallidurans carries two large plasmids: pMOL28 (171kb) and
pMOL30 (234kb) containing a variety of metal resistance genes (1).
The 19 genes of the pMOL30 cop cluster (providing a resistance to
copper) were sequenced, cloned in a broad host range vector and
reintroduced in the plasmid-free derivative AE104 where the wild-type
MIC was restored, the resulting strain being called AE1744. In this
study, flow cytometry was used to investigate the sensitivity to Cu++ at
sub-lethal concentrations on the three strains (CH34, AE104 and AE1744)
of C. metallidurans. To our knowledge, this work is one of the very first
flow cytometric studies of a bacterial resistance to a heavy metal. Size,
granularity, cell membrane permeability and potential and intracellular
esterase activity were chosen as good indicators of bacterial physiological
changes. Furthermore, the production of reactive oxygen species (ROS)
was also monitored since it is known that heavy metals generate free
radicals. This approach already allowed us to observe that cop plasmidic
genes reduce ROS level significantly, even at low (sub-lethal) copper
concentrations. Further investigation will now focus on the plasmidic
cop gene mutants and on the variations in the physiological response
measured by flow cytometry induced by low (sub-lethal) Cu++ in function
of each mutation S. van Aelst is supported by a doctoral FRIA grant.
1. M. Mergeay et al, FEMS Microbiology reviews, 26:385-410, 2004.
2. S. Monchy et al, submitted to Microbiology. 3. S. Baatout et al, Ann.
Microbiol., 55:73-80, 2005.
228/P82
FLOW CYTOMETRY TO ANALYZE BACTERIA MARKED
WITH FLUORESCENT PROTEINS
Nico Boon1, Arie Marel2
1
University of Gent, Gent, , Belgium; 2Dako, Flow Cytometry,
Heverlee, Belgium
Flow cytometry is an exceptional analysis tool for microbiology as
examplified by quantification of bacterial populations marked with
fluorescent proteins such as green fluorescent protein (GFP). One
interesting study subject is horizontal gene transfer for which plating
sometimes may be an inefficient tecnique. An example of GFP based
monitoration of transconjugation is shown. Finally, flow cytometry
allows for efficient sorting of baterial populations based on their
fluorescence profile. The highly purified sorted fractions are further
characterised by molecular analysis.
229/P83
ELECTROLYTIC DISINFECTION OF ESCHERISCHIA
COLI AND COLIFORM BACTERIA IN A BATCH CELL
WITH DSA ELECTRODES
Bradley JAY Hernlem1
1
Agricultural Research Service (ARS) - Pacific West Area,
Foodborne Contaminants Research, Western Regional Research
Center (Albany, CA), Albany, California
Electrolytic treatment of dairy manure lagoon water using DSA electrodes
is shown to produce a progressive disinfection of native and introduced
coliform and E. coli. The disinfectant effect continues post-treatment
for several minutes. To further examine the process, flow cytometry
was employed to study the disinfection of E. coli suspended in salt
solutions. Bacterial viability was estimated both by plate counting and
by the use of membrane integrity “viability” staining. The two methods
displayed proportional agreement. Furthermore, it was observed that
electrolytic treatment resulted in not only a decrease in the proportion
of putatively “live” cells but also in a reduction of overall bacterial
concentration, i.e. there appears to be a physical loss of bacteria as well
as degradation in cell viability. Plating and counting, alone, is not
sufficient to show this effect.
230/P84
QUANTITATIVE CHARACTERIZATION OF
PROTECTIVE ANTIGEN BINDING TO HUMAN TARGET
CELL TYPES
Alina Deshpande1, Claire Sanders2, Rebecca Hammon2,
Steven Graves2
1
Los Alamos National Laboratory, Decision Applications Division,
Los Alamos, New Mexico; 2Los Alamos National Laboratory,
Bioscience Division, Los Alamos, New Mexico
Using quantitative flow cytometry and fluorescently labeled Protective
Antigen (PA83), we have evaluated the binding of PA83 to human cell
lines that represent monocytes, epithelial cells, neutrophils, macrophages
and endothelial cells, The goal of this study was to determine the role of
both receptor affinity and receptor number in differential PA83 binding
to these cell types which are potential human targets for anthrax lethal
toxin. Regardless of cell type, we observed similar dissociation constants
in the low nM range. However, we observed increased numbers of PA
receptors on adherent cell types. Specifically, we observed a consistent
twofold increase in the number of surface receptors following the
differentiation of the monocytic cell line THP-1, which grow in
suspension, to the adherent macrophage phenotype. The same
phenomenon was also seen in HL-60 cells when differentiated to a
macrophage phenotype, but not when differentiated to a suspensiongrowing neutrophil phenotype. We propose that the differentiation
mediated susceptibility of monocytes to lethal toxin may be a result of
increased uptake of the toxin by increased numbers of receptors.
Furthermore, we observed high numbers of specific cellular receptors
for PA83 on endothelial cell types, as compared to monocytes, which
supports recent observations that lethal toxin (of which PA83 is the
binding element) at least partially targets the endothelium. Additionally,
epithelial cells also had a larger number of receptors as compared to
153
non-adherent cell types. The increased number of PA receptors on
adherent cell types suggests a role for PA receptors in interactions between
the cell and extra-cellular matrix and that PA binding targets lethal and
edema toxin to adherent cells.
231/P85
A DISSOCIATION AND STAINING PROCEDURE FOR
PARAFFIN-EMBEDDED TISSUES ENABLING FLOWSORTING OF NORMAL STROMAL CELLS AND
TUMOUR CELL SUBPOPULATIONS FOR FURTHER
MOLECULAR GENETIC ANALYSIS
Willem E. Corver1, Natalja Ter Haar1, Freek Blanken1, Anya
Milne2, Johan Offerhaus2, Tom Van Wezel1, Hans Morreau1,
Cees J. Cornelisse1, Gert Jan Fleuren1
1
2
Leiden University Medical Centre, Leiden, Netherlands;
Amsterdam Medical Centre, Pathology, Amsterdam, Netherlands
Contaminating normal stromal cells as well as lack of patient-matched
normal blood or tissue samples, frequently impairs accurate detection
of loss of heterozygosity (LOH) in archival paraffin-embedded tumour
tissues. We have developed a robust dissociation and three-colour
staining procedure for paraffin-embedded tissues that circumvents these
limitations by phenotypic identification and flow-sorting of keratinpositive tumour cells as well as vimentin-positive, keratin-negative
stromal cells (J Pathol. 2005 Jun;206(2):233-41). The procedure was
successfully applied to breast, cervical, colorectal and gastric cancer
archival tissues. High-resolution DNA histograms were obtained. DNA
extracted from the vimentin-positive, keratin-negative cell fractions
only showed retention of heterozygosity and could be used as an intrinsic
reference for the detection of LOH in tumour samples, without the need
of normal blood DNA from the same patient. Owing to the simultaneous
use of a DNA stain, the vimentin-positive, keratin-negative cell fraction
could be used as an internal DNA diploid reference. This allowed the
clear detection of DNA hypodiploid and hyperdiploid tumour cell
subpopulations in archival paraffin-embedded samples. This method
obviates the need for fresh / frozen tumour tissue for high-resolution
DNA ploidy measurements and facilitates molecular genetic analysis of
tumour cell subpopulations found in archival tumour tissues.
232/P86
MUTANT SPECTRA FROM GAMMA RADIATION AND
EMS MEASURED BY MULTICOLOR FLOW
CYTOMETRY
Stephen B. Keysar1, Carley D. Ross1, C. Tenley French2,
Michael H. Fox3
1
Colorado State University, Cell and Molecular Biology, Colorado
State University, Fort Collins, Colorado; 2Cytomation GTX, Fort
Collins, Colorado; 3Colorado State University, Radiological Health
Sciences, Colorado State University, Fort Collins, Colorado
Background: We recently developed a flow cytometry based mutation
assay to measure mutations in the CD59 gene on human chromosome
11, which is stably incorporated in a hybrid Chinese hamster ovary cell
line (CHO AL). Mutants are characterized by very low fluorescence
when stained with monoclonal antibodies specific for CD59, a surface
marker on CHO AL cells. Additional genes found on chromosome 11
(CD44 and CD90) are also expressed on the surface and can be used to
create mutant spectra for various genotoxic agents by using
multiparameter flow cytometry to measure all three antigens
simultaneously. CD59 is 1.4 Mbp from CD44 and 85.5 Mbp from
CD90. Methods: CHO AL cells were treated with ã-radiation or ethyl
methane sulfonate (EMS) and grown for various times to allow for
mutant expression. Cells were stained with monoclonal antibodies against
CD59. Single cells and populations of 500 to1000 cells were sorted
from four regions of the mutant peak and were expanded into stable
populations. Individual clones were labeled with PE-conjugated antiCD59 to measure relative fluorescence. Multicolor analysis of mutations
was done by staining cloned populations with antibodies specific for
CD59 (PE), CD44 (biotin-streptavidin Alexa 488) and CD90 (Alexa
647). Results: Simultaneous labeling for all three CD markers allowed
the analysis of mutations in all three genes after mutagenesis with different
agents. Double mutants in both CD59 and CD44 were common after
gamma radiation but rare after EMS. Triple mutants in CD59, CD44
and CD90 were much less common after gamma radiation and virtually
154
non-existent after EMS. Individual clones were isolated that were
mutated in all combinations of the CD markers so that PCR could be
used to validate the mutant spectrum. Conclusions: EMS and ã-radiation,
a point mutagen and clastogen generated very different mutant spectra
since they interact with DNA in different manners. CD59-/CD44- cells
are likely more common than CD59-/CD90- cells since large deletions
would encompass both CD59 and CD44 more often since they are
closer on chromosome 11 than CD59 and CD90. Multicolor analysis
of mutants with flow cytometry sheds light on the mechanism of mutant
induction from different genotoxic agents.
233/P87
THE ACE SYSTEM, A MAMMALIAN ARTIFICIAL
CHROMOSOME ENGINEERING TECHNOLOGY:
GENERATION OF A PLATFORM ACE HOST CHO CELL
LINE FOR HIGH-YIELD RECOMBINANT PROTEIN
PRODUCTION
G. Neil Mac Donald1, Sandra Babich1, Anne-Rachel
Fontanilla1, Alexisann Maxwell1, Diane Monteith1, Joan
Shellard1, Sharon Sidhu1, Malcolm Kennard1, Harry C.
Ledebur1
1
Chromos Molecular Systems Inc, Burnaby, British Columbia,
Canada
Mammalian artificial chromosomes provide an ideal means to introduce
large payloads of genetic information into a cell to rapidly engineer
mammalian cell lines to express recombinant proteins and monoclonal
antibodies at industrial levels. The ACE System consists of the Platform
ACE host cell line that contains a mammalian artificial chromosome
(Platform ACE) engineered with multiple site-specific integration sites,
expression-optimized ACE Targeting Vectors (ATVs) to specifically
transfer genes, and a proprietary integrase (ACE Integrase) to catalyze
specific incorporation of ATVs onto the Platform ACE. Once introduced
into a cell, ACEs are stably maintained, non-integrating, autonomously
replicating, are easily isolated to high purities by flow sorting, and can
be transferred to cells (including human stem cells) by lipid-mediated
ACE transfer. We describe the generation and characterization of a
CHO Platform ACE host cell line ChK2. The process consists of ACE
isolation by flow sorting, lipid-mediated ACE transfer into CHO cells,
and single-cell subcloning by flow sorting. The integrity of the ACEs
carried in ChK2 cells was determined by fluorescent in situ hybridization
(FISH) and flow cytometry. The stability of the Platform ACE was
monitored and found to be maintained in the host cell line for greater
than 20 passages. The ChK2 cell line was further characterized by
loading the Platform ACE with a monoclonal antibody (MAb) and
screened for high MAb expression. The ATV encoded both the heavy
and light chain MAb genes. Single-cell subcloned cell lines were
generated in less than 4 months and expressed MAb titres of > 400mg/
L in non-optimized 250ml batch shake flasks.
234/P88
VH GENE USAGE AND SOMATIC MUTATION
DISTRIBUTION CONSISTENT WITH ANTIGEN-DRIVEN
SELECTION IN BOTH ‘MUTATED’ AND ‘UNMUTATED’
CASES OF B-CELL CHRONIC LYMPHATIC LEUKEMIA
Karen Hensen1, Brigitte Maes1, Rita Smets1, An Broekmans1,
Sabine Franke1, Greet Bries2, Veerle Peeters1, Reinoud
Cartuyvels1, Jean-Luc Rummens1
1
Virga Jesse Hospital, laboratory of molecular biology and
experimental hematology, Hasselt, Belgium; 2Virga Jesse Hospital,
Department of Hematology, Hasselt, Belgium
In B-cell chronic lymphatic leukaemia (CLL), part of the cases shows
mutated VH genes indicating that the transformed B-cell has passed
through the germinal centre where it has undergone the somatic
hypermutation machinery during an antigen (Ag) response. In order to
examine the possible role of Ag stimulation in the pathogenesis of BCLL, we analysed 40 VH sequences derived from 37 CLL patients. VH
genes were amplified, sequenced and aligned with all known germline
VH genes available on the internet (IgBlast and IMGT). The observed
number of replacement (R) mutations within the complementaritydetermining regions (CDRs) and the framework regions (FRs) (ObsR
CDR and ObsR FR) were compared with the calculated expected numbers
(ExpR CDR and ExpR FR), taking into account the inherent replacement
susceptibility of CDRs and FRs. The probability (p-value) that scarcity
or excess of R mutations resulted by chance only was calculated for
CDRs and FRs using the binomial distribution model. VH gene usage
was biased with exclusive use of VH3 (20), VH4 (11) and VH1 (9)
genes. VH4-34 and VH3-30 genes were respectively 6 and 4 times used.
For 23 of 26 mutated VH genes (homology < 98 %), either the ‘ObsR
CDR´ was higher than the ‘ExpR CDR´ or the ‘ObsR FR´ was lower than
the ‘ExpR FR´ with p-values < 0,05 in 10 cases, indicating evidence for
positive and/or negative selection. Also one ‘unmutated´ VH sequence
showed evidence for Ag selection. The preferential usage of certain VH
genes as well as the skewed distribution of R mutations indicates that
certain Ags may be involved in the pathogenesis of CLL. The VH4-34
gene, most frequently used in this series, encodes for an auto-reactive
immunoglobulin (Ig) that is associated with B-cell lymphotropic viruses,
particularly EBV. Further studies of the binding sites of restricted Ig, are
necessary to elucidate the possibility of Ag involvement in CLL
development. Furthermore, as evidence of antigen selection is detected
in both ‘mutated´ and ‘unmutated´ CLL cases, its role in predicting
prognosis, in addition to the VH-mutation status, should be investigated.
235/P89
DETECTION OF CHROMOSOMAL ABNORMALITIES BY
INTERPHASE FLUORESCENCE IN SITU
HYBRIDISATION ON FLOW SORTED PLASMA CELLS
Karen Hensen1, Hanne Jongen1, Sabine Franke1, Veerle
Peeters1, Brigitte Maes1, Jean-Luc Rummens1
1
Virga Jesse Hospital, laboratory of molecular biology and
experimental hematology, Hasselt, Belgium
Multiple Myeloma (MM) is a malignant clonal neoplasia of plasma cells
commonly resulting in overproduction of monoclonal immunoglobulins.
The plasma cells are phenotypically characterised by a strong expression
of CD38 and CD138 but can display an aberrant phenotype compared
to normal plasma cells. Besides other markers, asynchronous expression
of CD56 is reported in the majority of MM patients. Cytogenetic
abnormalities, mostly evaluated by conventional techniques like
karyotyping and fluoresence in situ hybridisation (FISH), are considered
to be the most important prognostic markers of poor prognosis in MM,
such as changes in chromosome 13q and translocations of 14q32.
However the detection and characterization of these genetic aberrations
is often hampered by the low proliferative index of plasma cells, the
limited extent of bone marrow (BM) involvement or the limited
proportion of cells bearing the abnormality. In order to overcome these
difficulties, we optimised a combined protocol of fluorescence activated
cell sorting (FACS) of clonal plasma cells followed by subsequent
interphase FISH analyses, using a broad panel of probes. Plasma cells of
MM patients at various stages of treatment and disease were purified
based on the clonal expression of Ig light chains and CD56 within the
CD138+/CD38++ plasma cell gate. A purity between 90-95% of the desired
population could be obtained as measured by flow cytometry and cell
morphology. In these purified cell populations, several chromosomal
aberrations could be detected, with the most frequent aberrations being
del13q, IGH rearrangement, gain of 1q, trisomy 19, monosomy 14,
gain of 19p and loss of 19q. For all cases, at least one genetic change
could be identified in the sorted cells. Abnormalities found in these
sorted plasma cells were undetectable on the corresponding smears.
The percentage of cells bearing the abnormality varied from 10-80%.
These data clearly demonstrate that interphase FISH performed on FACS
sorted plasma cells has a high sensitivity for the detection of chromosomal
abnormalities. Its application has revealed a higher prevalence of
chromosomal abnormalities in MM compared with classical methods.
Moreover, within phenotypically pure MM cell populations, more than
one genetic clone seems to be present. Furthermore, the fact that
suspensions of sorted plasma cells are suitable for further cytogenetic
and molecular analyses, could be particularly important for more
sensitive techniques like micro-array analysis in which a pure
homogenous sample is highly desirable.
236/P90
A SEMI DOUBLE EMULSION PROCESS FOR
RESERVOIR-TYPE MICROCAPSULES GENERATION
Sang-Youp Lee1, Connie Snider2, Kinam Park3, J. Paul
Robinson4
1
West Lafayette, Indiana; 2Purdue University, Industrial and Physical
Pharmacy, West Lafayette, Indiana; 3Purdue University,
Pharmaceutics and Biomedical Engineering, School of Pharmacy,
West LaFayette, Indiana; 4Purdue University, Basic Medical
Sciences & Biomedical Engineering, West Lafayette, Indiana
A novel method for micro-scale aqueous droplet encapsulation with
poly(lactide-co-glycolide) (PLGA), a biodegradable polymer, has been
designed based on flow cytometry technology. In this technique, the
double emulsions are produced in two steps as in the conventional
double emulsion process. The conventional method relies on the
mechanical agitations of the massive fluids resulting in the large amount
of waste of materials and wide (or random) size distribution. The present
technique, however, places controls on the size distribution by utilizing
an experimental component, termed as emulsion chamber, where the
primary emulsion is generated using an ink-jet nozzle. The major
advantages of the emulsion chamber setup is that the complicated physics
among multiple fluids can be simplified by adopting the two-step process
and more precise size control on the aqueous core is available due to the
well-defined ink-jet nozzle fluidics. The primary emulsion is fed into a
stream of the third aqueous medium, i.e. sheath flow. The second
emulsification step is performed while the primary emulsions are
separated into pieces of desired small volumes within the stream. During
the secondary emulsification, the precipitate process, known as solvent
exchange, takes place to form a polymer membrane around the aqueous
core. The experiment was performed with 2% (w/v) PLGA solution in
ethyl acetate. An ink-jet nozzle with a diameter of 40~60µm dispenses
the monodisperse aqueous droplets over the polymer solution bath
within the emulsion chamber at the frequency range of 9.7~9.9 kHz
and with an amplitude of 100 mVpp. Because the density of aqueous
droplets is greater than that of polymer solution, the aqueous droplets
travel downward within the polymer solution. The emulsion chamber
pressure forces the primary emulsion into the third water stream, where
the precipitation happens, through a needle. The stream of third medium
is confined with an orifice 100~150µm in diameter and the polymer
membrane thickness is controlled by adjusting the differential pressure
between the emulsion and flow chambers. Microcapsules are observed
using both light and fluorescent microscopes. Preliminary results show
that the semi double emulsion setup successfully generates reservoirtype mononuclear microcapsules in the range of ~20 µm. While we
have made successful mononuclear encapsulations, there are still a
number of issues to solve regarding size distribution. The observed
cause of nonuniformity of aqueous core is likely due to the coalescence
of aqueous cores in the emulsion chamber and, in order to stabilize the
primary emulsion in the emulsion chamber, various surfactants are
being tested.
237/P91
MICROFABRICATION AND CONSTRUCTION OF A
BIOMEMS MICROFLUIDIC CELL SORTER
Meggie Grafton1, Lisa M Reece2, Kwanseop Lim3, Yi-Shao
Liu4, Rashid Bashir5, James F. Leary2
1
Indiana; 2Purdue University, Basic Medical Sciences, West
Lafayette, Indiana; 3Purdue University, ECN, West Lafayette,
Indiana; 4Purdue University, West Lafayette, Indiana; 5Purdue
University, School of Electrical and Computer Engineering,
Closed system, microfabricated BioMEMS (Bio
MicroElectroMechanical Systems) microfluidic cell sorter devices would
represent a major advance for cell sorting, especially in terms of
providing an aerosol free means of sorting potentially biohazardous
materials within a standard biosafety hood.
A microfluidic sorter
BioMEMS was fabricated and tested. The microfluidic portion consists
of a disposable PDMS ((poly)dimethylsiloxane)) biochip sandwiched
between light sources and photodetectors. The entire multicomponent
device is being systematically miniaturized. Laser light sources are being
replaced by inexpensive, battery-powered, superluminescent light
155
emitting diodes (LEDs) and photomultiplier tubes are being replaced
by avalanche photodiodes (APDs).
The National Instruments (NI)
data acquisition system consists of a PXI-8187 embedded controller, speed NI PXI-6281 M-series multifunction DAQ boards all running
under National Instrument’s LabView Realtime 7.1. The sort logic
created in the NI-LabView system controls elastomeric valves within
the PDMS microfluidic chip.
238/P92
INTEGRATION OF MULTIPLE HIGH-SPEED DROPLET
CELL SORTERS INTO A STANDARD BIOSAFETY
CABINET IN A MANNER THAT PROVIDES BSL-2
CONTAINMENT
shown that this new fluidic sub-system completely recapitulated the
intended function of the manufacturer’s standard fluid handling system,
and isolates the fluid from contaminants such as bacteria and fungus,
endotoxins, mycoplasma and helicobacter. Classical mouse hematopoietic
CFU assays performed demonstrated similar colony forming efficiencies
when unsorted serially diluted samples were compared to samples
prepared using cells sorted with our modified instrument. FACS has
emerged as a powerful tool used to study and manipulate stem cells.
However, if stem cell discoveries are to be fully utilized in clinical
transplant medicine, aseptic instrument configurations must be
developed. For this purpose, we have designed a sterile fluid handling
system that is autoclavable in the research setting and has potential to be
mass produced as a disposable flow cytometer component for clinical
applications.
Gary Durack1, Paul Weiss1
1
iCyt Visionary Bioscience, Champaign, Illinois
We have successfully integrated two high-speed, droplet cell sorters
into a single 48" wide biosafety cabinet (BSC). The components of the
cell sorters contained in the BSC are the sample delivery system, nozzle,
deflection plates, sheath delivery system and sort collection system. All
fluid delivery, sample delivery, sort collection and waste collection are
contained in the BSC. All lasers, laser steering optics, photodetectors,
pneumatic components and electronic components are located outside
of the BSC. Sealed pass-thru conduits route all pneumatic tubing and
electronic cables through the wall of the BSC. When a sample is running,
the sorter stream paths inside the BSC are completely sealed to prevent
the possibility of aerosol escape into the BSC. This is accomplished by
insertion of all sample and collection tubes into specialized cassettes that
seal access to the droplet stream when inserted into the sorter. Sensors
located in the cassettes prevent sample flow when the cassettes are not in
place. This eliminates the possibility of contamination when tubes are
removed. BSL-2 compliance is achieved by locating these completely
sealed stream paths inside the BSC, thus providing two barriers to protect
both the sample and the operator. The optical alignment, droplet breakoff control and sort calibration are completely automated and do not
require routine user intervention inside the BSC. Data were collected
from numerous sorting experiments. Several fault conditions were
simulated, such as nozzle clogs, sample line leaks and collection tube
overflow. Results of the sorting experiments show that even in the case
of the most severe failure modes, no contamination from the sorter
could be detected inside the BSC. This integrated system provides a
sorting capacity of 140,000 cells per second in a standard BSL-2
compliant BSC.
239/P93
STERILE AND DISPOSABLE FLUIDIC SUB-SYSTEM
SUITABLE FOR CLINICAL HIGH SPEED FLUORESCENT
ACTIVATED CELL SORTING
Sach Jayasinghe1, Joshua Wunderlich1, Angel McKee1,
Heather Newkirk2, Linheng Li1, Karen Staehling-Hampton1,
Steve Pope3, Jeffrey S Haug1
1
Stowers Institute for Medical Research, Kansas City, Missouri;
Children’s Mercy Hospital, Kansas City, Missouri; 3R2FACT
Design & Engineering, Inc., Lenexa, Kansas
2
Applications of FACS are ideally performed under aseptic conditions so
that isolated cells can be successfully cultured, transplanted, or processed
for the isolation of protein and nucleic acids. However, modern “offthe shelf” flow cytometers are sub-optimally designed for these purposes
because non-sterile instrument hardware components directly contact
sample harboring fluids, compromising their sterility. The use of in-line
micro-pore filters and cleaning reagents are still needed to condition the
inner lumens of the fluidic sub-systems. As such, the current FACS
technology, at best, will provide cellular products that have contacted
the inner lumen of an instrument that has been exposed to foreign
cellular products and adventitious reagents such as bleach or ethanol.
We have described the design and modular modification of a flow
cytometer with a sterile and disposable FACS fluid handling system that
meets requirements of high-speed FACS and GMP. This fluid sub-system
has multiple elements: 1) a GMP quality sheath fluid bladder pressurized
in a tank, 2) autoclavable or disposable tube set with sterile terminations
and no in-line valves, 3) a tube set mounting plate with pinch valves, 4)
autoclavable backflow protected waste valves, and 5) a disposable or
autoclavable flow nozzle. This system was tested for functionality and
its ability to maintain a clean and sterile fluid environment. Our data has
156
Autoclavable Flow Nozzle Design
240/P94
REMOTE-CONTROLLED BIOHAZARD CELL SORTING
Kenneth Ketman1, Natasha Barteneva2
1
CBR Institute for Biomedical Research, Boston, Massachusetts;
CBR Institute for Biomedical Research, Harvard Medical School,
Department Pathology, Boston, Massachusetts
2
The importance of flow cytometry sorting has grown dramatically with
a significant increase in sorting applications used for immunology and
molecular biology. Contemporary jet-in-air cell sorters, as a part of
their normal operation generate droplets that can be aerosolized.
Biohazard sorting is not limited to the samples containing infectious
agents but applies to sorting of unfixed cells of any origin that may
carry pathogenic organisms known to infect humans (Schmid et al,
2004).
To minimize exposure to pathogenic microorganisms and
viruses, it is necessary to perform the cell sorting with adequate protection
of cell operator. The measurement of aerosol protection in cell sorting
recently done with GloGerm beads and particle counter or air sampler
under conditions imitating 25 psi pressure (Oberyszyn, Robertson, 2001;
Perfetto et al, 2003; 2004). However, system pressure achieved at
contemporary high-speed sorters far exceeds 25 psi. Even well-operated
biological safety cabinets lose a small fraction of aerosolized particles.
Various types of respirators differ in their predicted efficiency and
ability to reduce the risk aerosol inhalation with even full-face piece
powered air-purifying respirator not giving 100% protection of
personnel. Therefore, a successful strategy for cell sorting was
developed that allows remote-controlling cell sorting. In establishing
this new technique, we have used FACSAria cell sorter (BD Biosciences,
San Jose, CA), which was installed inside walk-in Baker BioProtect II
biological safety enclosure, remote computer station, video cameras
and telemonitors. Video cameras were located inside biosafety cabinet
and allowed to watch level of liquid in collection and sample tubes.
Operationally, the remote-controlled computer connected with FACSAria
started together with cell sorter. We have further tested our technique
with various types of multicolor sorts. Regardless of performed 2- or 4way sorts, we were able to control sorting from remote computer station.
Our laboratory is routinely doing cell sorting of HIV-infected cells, as
well as other types of biohazard sorting (parasites, lentivirus-transduced
cells etc). Introduction of this technique allows sorting cells without
physical presence of cell operator in cell sorting room during biohazard
sort, i.e. when aerosolization of sorting sample may happen. We are
confident that this technique will be embraced by many cell operators
working with biohazard samples or even simply willing to operate
FACSAria together with other instruments in a walk-away mode. The
potential for remote controlling extends beyond silent monitoring of
sorting events on neighboring sorter. The technology in combination
with robotic arm can be safe solution for biohazard sorting.
241/P95
LARGE PARTICLE SORTING WITHOUT A NOZZLE ON
THE BD FACS ARIA
David Houck1
1
Becton Dickinson Biosciences, San Jose, California
With jet in air sorting, it is often possible to adjust the nozzle size to
optimally sort particles of different sizes. Initially the sorters were
developed to sort lymphocyte subpopulations with 50 and 70 micron
nozzles and then large particle sorting was developed with nozzle sizes
up to 400 microns. The FACS Aria nozzle and flow cell combination
were dimensioned to support lymphoctyte and large tissue culture cell
sorting with nozzles of 70 and 100 microns. It is possible to remove the
nozzle on the Aria and still generate a jet from the 160 X 250 micron
flow channel and make droplets to facillitate sorting of larger particles.
Limitations on particle size and sort rate will be discussed and results
from examples of large particle sorting will be shown.
242/P96
LARGE PARTICLE FLOW CYTOMETRY FOR STUDY OF
CELL CLUSTERS
Rock Pulak1, Bo Wang1, Julia Thompson1, Rico Bongaarts2
1
Union Biometrica, Inc., Holliston, Massachusetts; 2Union
Biometrica, Inc, Geel, , Belgium
We describe applications for analysis and sorting of intact pancreatic
islets and EBs using the large particle flow cytometer, the BioSorterTM
1000. BioSorter instruments can sort large (40-1500 micron) particles
in a continuously flowing stream at a rate of 10 - 50 objects per second.
Using object size, optical density, and intensity of fluorescent markers
as sorting criteria, particles can be dispensed into Petri-dishes or multiwell plates for further analysis. A gentle pneumatic sorting mechanism
located after the flow cell does not harm or change sensitive objects,
thereby making the instrument suitable for live biological materials or
sensitive chemistries. Multiple fluorescence excitation and emission
wavelengths have been tested. For these experiments we used a three
laser configuration (325 nm UV, 488/514 nm multi-line Argon and 670
nm diode laser). Post analysis of intact cell clusters (islets and EBs) run
through BioSorter 1000 flow cytometer has shown that viability and
functionality are unaffected. Preliminary tests using the BioSorter 1000
for several types of cell clusters, including analysis and sorting of intact
mouse, rat, pig, and human islets, as well as mouse EBs, have been
performed. We believe that studying intact cell clusters, rather than
disrupted single cells, should provide a better reflection of their normal
biology.
243/P97
OPTIONS FOR A HIGH-SPEED PHOTODAMAGE CELL
SELECTION USING GOLD NANOPARTICLES AND
PULSED LASER IRRADIATION
Florian Levold1, Sebastian Ziewer1, Franziska Winter1,
Gereon Huettmann2, Johannes Gerdes3, Elmar Endl1
1
University of Bonn, Bonn, Germany; 2Institute of Biomedical
Optics, Luebeck, Germany; 3Research Center Borstel, Borstel,
Germany
The interaction of pulsed laser irradiation with nanoparticles on cellular
surfaces can be used to effectively eliminate cells with high precision
and at high-speed. Cells can be inactivated by the use of gold as inorganic
absorber particles coupled to the target via cell type specific antibodies.
The particles itself act as well confined heat sources after irradiation
with Q-switched and mode–locked lasers and induces lethal membrane
damage to target cells whereas control cells are unaffected. We could
demonstrate that the damage mechanism is based on physical parameters
and can therefore be varied more precisely than chemical methods by
choosing appropriate particle size, pulse width and pulse energy.
Optimization of parameters lead to a reproducible efficiency of more
than 96% selective cell killing for various primary and cell culture cells
after a single pulse exposure to laser light. We also could show, that
eliminating subpopulations of cells from blood samples leaves basic
immunological functions of non target cells unaltered. In conclusion,
the efficient and precise destruction of cells by interaction of gold
nanoparticles and pulsed laser irradiation, for example in a fly-by mode,
might revitalize the idea of a photodamage cell sorter. These type of
sorter was suggested as a high-speed alternative to droplet sorter, more
than two decades ago, since there are minor limitations due to droplet
formation frequency or particle size. Moreover there is also no need for
the identification of target cells by analytical fluorescent techniques
since the targeting is achieved by appropriate coating of the nanoparticles.
Laser induced, nanoparticle mediated elimination may therefore
represents a new contact-free and high throughput method to effectively
remove contaminating cells from fragile cell populations without
affecting yield and viability of requested cell type.
244/P98
IMPLEMENTATION OF A FREQUENCY BASED METHOD
FOR SORTING IN FLOW CYTOMETRY USING XML
Geoffrey W. Osborne1, Geoffery Ericksson2
1
University of Queensland, Flow Cytometry, Queensland Brain
Institute, Brisbane, Queensland, Australia; 2University of
Queensland, Queensland Brain Institute, Brisbane, Queensland,
Australia
Traditionally, the selection criteria for cells/particles for separation are
user defined regions of interest drawn on one or more bivariate displays
of multiparametric data. Regions are defined using prior knowledge
regarding the characteristics of the particle of interest. The method we
describe requires no such prior knowledge for the setting of sort criteria.
Using this method, sorting is a function of the frequency of occurrence
of measured values in multidimensional space. This method is ideally
suited to samples where the flow cytometric phenotype is poorly known
or uncharacterised, yet the frequency of particular subpopulation is
known. It is thus applicable in tasks as diverse as sample decontamination
to rare event sorting, or conversely every combination event sorting. As
an example, this method can be used in conjunction with hierarchical
gating approaches to drive enrichment strategies for the selection of
stem cells. Computationally, this method is shown to effectively elucidate
the multiparametric phenotypic characteristics of both rare and plentiful
cell populations. Here we outline one method of implementing our
frequency based approach by generating XML files containing the
appropriate gates to define populations with the requested frequencies,
then importing these files into commercial flow cytometer software and
sorting on this basis.
245/P99
ACOUSTIC TECHNIQUE FOR ULTRASONIC PARTICLE
CONCENTRATION FOR UNIQUE FLOW CYTOMETRIC
ANALYSIS
Gregory Goddard1, John C Martin1, Steven W Graves1,
Michael D Ward1, Gregory Kaduchak1
1
Flow cytometers have applications in many areas including medicine
(HIV and cancer diagnosis), homeland defense (biological point
detection, bio-surveillance, and forensic analysis) and general biomedical
research (ligand-receptor studies, molecular assembly analysis, high
throughput screening and genotyping). Though hydrodynamic focusing
is highly effective at particle positioning, the use of sheath fluid increases
assay cost and reduces instrument utility for field and autonomous
remote operations. Although conventional flow cytometry is well adapted
to high throughput applications, hydrodynamic focusing limits the
volumetric flow rate, and thereby the ability to analyze highly dilute
samples. The present research investigates an alternative acoustic particlepositioning device for use in flow cytometry wherein acoustic excitation,
generated along the entire structure of a capillary tube, drives sample
particles to the central axis. Our technique will allow the creation of a
sheathless portable flow cytometer capable of conventional particle
analysis rates, while using a fraction of the power and consumables of
a conventional flow cytometer. Because acoustic methods both focus
and concentrate particles, it is possible to maintain both conventional
particle analysis rates as well as long transit times. The longer integration
time allows conventional particle analysis rates using data acquisition
systems that are less expensive, smaller and require less power, while
still performing high sensitivity measurements. However, the benefits
of acoustic focusing flow cytometry are not restricted to only the
elimination of sheath fluid. Acoustic concentration also enables the
analysis of extremely dilute samples on the order of several cells or
157
particles per liter, as might be seen in a water monitoring application, at
reasonable analysis rates. Since there is no sheath, it is also possible to
repeatedly reanalyze particles of interest for reliable rare event analysis.
Results of a series of demonstration experiments will be presented. This
work was supported by NIH RR020064-01, NIH RR001315-23 and
DOE LDRD funding.
248/P102
ANALYSIS OF HOECHST SIDE POPULATION (SP) CELLS
IN MOUSE BONE MARROW USING LOW-POWER UV
SOURCES
William G. Telford1, Ella G Frolova2, Raquel Cabana3,
Richard A. Thomas3, Awtar Krishan4
1
246/P100
ON-CHIP NON-INVASIVE AND LABEL-FREE CELL
DISCRIMINATION BY IMPEDANCE SPECTROSCOPY
Marco Di Berardino1, Grit Schade1, Adrian Huwiler1, Xavier
Houriet1, Thomas Hessler1
1
Leister Process Technologies, Axetris Microsystems Division,
Kaegiswil, , Switzerland
Various flow-cytometric cell characterization methods require costly
markers and colour reagents. We present a novel discrimination method
for cells based on the measurement of electrical cell properties in a
microfluidic chip without the need of extensive sample preparation
steps and the requirement of labelling dyes. Impedance measurements
provide information on size, membrane capacity and cytoplasm
conductivity of single cells. Combining this with dielectrophoresis as a
gentle cell handling technology non-physiological conditions can be
overcome, offering at the same time the possibility to supply the device
with a sorting functionality .We demonstrate that in-flow single cell
measurements in our microchip allow for discrimination of various cell
line types, such as undifferentiated mouse fibroblasts (3T3) and
adipocytes on the one hand, or monocytes and monocytes that
differentiated to dendritic cells and macrophages on the other hand. In
addition, the ability to separate blood cell types, e.g. lymphoblasts,
granulocytes and monocytes, emphasizes the suitability of the system
for many other haematological applications. For some cell models even
viability and apoptosis analyses were carried out successfully. Finally,
studies on several species, from bacteria to fungi not only demonstrate
the capability to enumerate these cells, but also show that even sporulation
and other microbiological live cycle phases can be visualized. These
results underline the potential of Impedance Spectroscopy Flow
Cytometry as a valuable complement to the known cytometers and
other cell detection systems.
247/P101
A NOVEL FLAT-TOP BEAM SHAPER AND ITS
APPLICATION TO FLOW CYTOMETRY
Yong Q. Chen1
1
A novel flat-top beam shaper and its application in flow cytometry will
be presented. Unlike conventional beam shapers, the current technology
is able to transform incoming laser beams of multiple transverse modes
at multi-wavelengths into a flat top with near 100% efficiency. A
backward compatible near-UV excitation source is developed for the
BD flag-ship high-speed sorter (Aria). Comparison to conventional
Gaussian beams, the flat-top technology offers a 10X increase in sample
throughput without compromising system CV. Application to stem-cell
side population studies will be presented and its benefits over traditional
UV excitation sources will be discussed.
158
National Institutes of Health (NIH), Experimental Transplantation
and Immunology Branch, National Cancer Institute (NCI),
Bethesda, Maryland; 2National Institute of Child Health and
Development - NIH, Laboratory of Mammalian Genes and
Development, Bethesda, Maryland; 3NPE Systems, Inc., Pembroke
Pines, Florida; 4University of Miami, Radiation Oncology, School
of Medicine, Miami, Florida
Discrimination of stem cells using flow cytometric analysis of Hoechst
33342 efflux by the ABCG2 transporter (termed the Hoechst “side
population”, or SP technique) is a valuable methodology for identifying
bone marrow progenitor enriched for stem cells. Unfortunately, it requires
a UV laser source, usually necessitating a large water-cooled ion laser or
an expensive solid state solid state UV source on a large-scale cell sorter
or benchtop analyzer. In previous work, we have demonstrated the
utility of low-power near-ultraviolet laser diodes (NUVLDs) in analyzing
the SP population on both cuvette and epifluorescence-based flow
cytometers. In this study, we have experimentally determined the
minimum UV power level required for the detection of the SP population
on both types of cytometers using NUVLDs with a range of power
levels, and using high-power UV-emitting LEDs in the epifluorescencebased system. A fiber-coupled NUVLD emitting at less than 3 mW gave
adequate excitation for detection of SP on both cuvette and
epifluorescence systems, and a focused UV LED gave resolution on the
epifluorescence-based instrument approaching that of laser sources.
These studies suggest that low levels of UV excitation, and non-coherent
sources of UV light are useful for SP detection, permitting the design of
low-cost analysis systems capable of analyzing this critical parameter.
249/P103
MEASURING SIZE USING FORWARD ANGLE LIGHT
SCATTER IS NOT STRAIGHT FORWARD
Murugesan Venkatapathi1, Gérald Grégori2, E. Dan
Hirleman3, J. Paul Robinson4
1
Purdue University, Mechanical Engineering, Schools of
Engineering, West Lafayette, Indiana; 2Université de la
Méditerranée, Marseille, France; 3Purdue University, Mechanical
Engineering, west lafayette, Indiana; 4Purdue University, Basic
Medical Sciences & Biomedical Engineering, West Lafayette,
Indiana
In flow cytometry the forward (FALS) and right (RALS) angle light
scatter intensities are two basic parameters that can be measured in most
of the commercially available flow cytometers. Though FALS intensity
is related to the size of the particle, it has been primarily used for
detecting every single particle flowing in the instrument with or without
natural or induced fluorescence. This is because the relationship between
the size and the forward scatter intensity is complex because it depends
also on several other variables like the refractive index, shape of the
particle, and the angular span of the scatter measurement. It is well
known that FALS and Right angle light scatter (RALS) do exhibit some
correlation with the size and refractive index of particles. But the exact
study of this correlation has been impeded by the analytical difficulty
of modeling scatter from a particle due to an incident Gaussian laser
beam (plane wave scatter models can be used only when laser beams are
much larger than particles) and the interactive scatter between particles
and flow channel. The aim of this work was to correlate particle size
with the FALS intensity obtained both by electromagnetic scatter models
and by experiments on a flow cytometer. The analytical scatter models
assume the particles are in isolation in the sheath fluid and the angular
scatter distribution is calculated and integrated over the area of the
forward scatter detector (most often a photodiode) placed outside the
sheath fluid. A plane wave approximation of the incident beam has
been used to take into account the Gaussian distribution in intensity of
the incident beam. Light scattered by several microsphere solutions of
different diameters has been studied and the implications for using
forward scatter for estimation of size of cells discussed as well. As has
been shown both theoretically and experimentally in this paper, the
convention that larger particles have a larger intensity of light scattered
into the forward scatter detector is not always true. Since the mask/beam
dump used to protect the forward scatter sensor from the laser beam
plays a crucial role in limiting the angles (span of measurement) at
which scatter is measured, two different measurement spans are measured
and analyzed. Also included is a similar comparison between the
analytical relationship between size and total scattered energy collected
(integral of intensity over time of flight of particle). The conclusion of
this study is that angle resolved (multiple angle) scatter measurements
are required to correctly estimate size of particles in a flow cytometer.
250/P104
IMPROVED FORWARD SCATTER DETECTOR FOR
FLOW CYTOMETRIC CHARACTERIZATION OF SMALL
PARTICLES
Timothy Petersen1, C. Brent Harrison1, Jarred Swalwell2, Ger
Van Den Engh3
1
Cytopeia, Inc., Seattle, Washington; 2University of Washington,
Department of Oceanography, Seattle, Washington; 3University of
Washington, Genome Sciences, Seattle, Washington
Flow cytometry has been used successfully to study microbes for over
twenty years. While many of these studies have used custom-built or
commercial instrumentation, few commercial instruments have been
modified and optimized to detect very small organisms. We modified
the forward scatter optics of a commercially available cell sorter to
increase its ability to detect small particles. In addition, both the forward
scatter and side scatter detectors were constructed to measure two
orthogonal polarizations, a technique that may prove useful for detecting
calcite-containing plankton such as coccolithophores. A third
modification was the use of a red-sensitive Peltier-cooled PMT for
measurements of chlorophyll. The modified sorter was tested using
beads and both axenic cultures and natural samples from the Bermuda
Atlantic Time Series (BATS) and concentrated samples from Chesapeake
Bay. This presentation details the extent of the improvements that we
were able to document and their utility for marine picoplankton analysis.
251/P105
LIGHT SCATTERING: STATE OF THE ART AND FUTURE
IN IDENTIFICATION AND CHARACTERIZATION OF
CELLS
Valeri P. Maltsev1
1
Institute of Chemical Kinetics and Combustion, Novosibirsk,
Novosibirsk, Russia
Light scattering plays a weak role even in identification of cells because
of complexity of this phenomenon in theory and interpretation. Flow
cytometric forward and side light-scattering signals form the insufficient
set of informative parameters to improve recognition of cells. However
the multiangle light-scattering technique that is only available in
instrument developing laboratories demonstrated substantial
enhancement in identification of cells from light scattering. Scanning
Flow Cytometry (SFC) allows measurement of angular light-scattering
profiles of individual cells within the region ranging from the forward
to side direction. As an example the decision function created from the
standard fluorescence-based analysis was used to identify T and B
lymphocytes from multidimensional normal distribution of lightscattering parameters measured with SFC. Moreover physical cellular
characteristics such as cell volume, cell shape, nucleus volume, density
of nucleus and cytoplasm can be determined from light scattering in
flow cytometric analysis providing characterization of cells. The
determination based on the original solutions of the inverse lightscattering problem for particles with complex shapes and structures. At
present the following cells can be effectively characterized from light
scattering: mature red blood cells, blood platelets, monocytes,
lymphocytes, rod-like bacteria. Characterization of cells plays an
important role in development of quantitative instrumental technologies
for predictive biology. Polarizing effects in a light scattered by cells
form the outstanding potential of light-scattering technologies for
identification and characterization of cells. The fist experiments with
polarizing multiangle light-scattering profiles were carried out measuring
mature red blood cells and progenitor cells with the polarizing SFC.
These experiments are prerequisite in development of the light-scattering
tomography. Modern optics and mathematics are able to provide
reconstruction of 3-dimensional image of a cell with nanoscale resolution
from polarizing light scattering. This reconstruction can be realized for
cells undamaged by fluorescent dyes used for 3D reconstruction in
laser scanning microcopy.
252/P106
COMPUTER-SUPPORTED QUALITY CONTROL IN DNA
FLOWCYTOMETRY ANALYSIS
Nick Van Rodijnen1, Eric Postma2, Sjack Hoop1, Mathy
Leers3, Marius Nap1
1
Atrium Medical Center, Heerlen, Limburg, Netherlands;
University Maarticht (UM), IKAT, Maastricht, Limburg,
Netherlands; 3Atrium Medical Center, Heamatology, Heerlen,
Limburg, Netherlands
2
Background The essence of quality control (QC) is the presentation of
evidence that a process was performed according to the documented
protocol. The process of analysis of flowcytometric DNA histograms is
based on protocols that are applied by human operators, which may
lead to non-consistent ploidy labelling. Automation of this process will
contribute to the reliability of the final classification. The research
question addressed in this paper is; to what extent is it possible to develop
a procedure for automated QC and analysis of manually-labelled
flowcytometry DNA histograms. Methods We present an algorithm that
models a flowcytometry dataset and maps all individual DNA histograms
on a standardised neutral interpretation axis (NIX), after a linearity
check and diploid G0/G1 peak analysis. The NIX mapping of
flowcytometry DNA histograms minimises the variation between
histograms by correction for drifting. This enables cross comparison of
all DNA histograms in potentially large datasets. The algorithm counts
the number of pre-defined classification characteristics present in the
mapped DNA histograms to arrive at a ploidy classification. To evaluate
the algorithm we submit 833 manual-labelled DNA histograms, which
might contain errors due to inconsistent labelling. Our algorithm is
effective if it can detect low quality DNA histograms, detect inconsistent
labelling of the DNA histograms in the dataset and present a label
suggestion. Results In our dataset of 833 DNA histograms, the algorithm
rejected 4% (33 cases) of the DNA histograms based on non-linearity,
in 0,6% (5 cases) of the histograms the wrong diploid G0/G1 peak was
detected, and in 1% (9 cases) no mean S-phase height could be calculated.
In addition we found 11% (94 cases) with a skewed diploid G0/G1
peak, which were not automatically analysed. In the remaining cases an
automatic suggestion for classification could be given, with direct visual
inspection on screen for acceptance or rejection. Reclassification from
diploid to tetraploid is suggested for 13 cases and reclassification from
tetraploid to diploid in 51 cases. Conclusion Standardisation of
flowcytometry DNA histogram representation with the NIX mapping is
an important condition for automated quality control of the manual
ploidy labelling. We argue that the adaptation of the NIX mapping
allows for the cross-laboratory integration of histogram datasets.
253/P107
AUTOMATED FLOW CYTOMETRY FOR STUDYING
TIME DEPENDENT CELL PHENOTYPES
Ann Hansgate1, Alan Gilbert1, Greg Sitton1, Friedrich Srienc1
1
University of Minnesota, CEMS and BTI, St. Paul, Minnesota
Flow cytometry analysis usually requires careful preparation of the cell
samples involving cell fixation, washing, and staining operations.
Traditionally these steps are carried out manually restricting the number
and reproducibility of samples that can be analyzed.
However, for
investigations of growing cells that change in time, it is important to
follow the population dynamics over extended time periods with frequent
cell sampling. To accomplish this we have developed an automated cell
preparation device that interfaces with the cell culture vessel and a flow
cytometer allowing cell sampling and analysis on a time scale of every
few minutes. The instrumentation can dilute or concentrate cells to the
optimum cell concentration for best analysis and is able to carry out
common cell staining operations. Moreover, the automated analysis
approach permits implementation of a feed back control strategy that
controls the condition of the cell culture as a function of measured cell
properties. We have applied this approach to monitor and control batch
and continuous bacterial, yeast and mammalian cell cultures. The
obtained batch cultivation data represent a detailed, highly reproducible
fingerprint of time dependent cell characteristics as a function of
cultivation conditions. In continuous cultivation experiments a precise
connection between cell dynamics and environmental conditions can
be determined because a steady state in both cell population and cell
environment can be established. The evaluation of a cell phenotype at
this level of detail and precision becomes increasingly important in the
attempt to identify a valid link between cell function and the information
available at the genomic level.
159
254/P108
INFLUENCE OF FLOWCYTOMETERS AND
ACQUISITION/ANALYSIS SOFTWARES ON
DETERMINATION OF LYMPHOCYTE SUBSETS IN HIV
INFECTION
Deshratn Asthana1, Margarita Ashman1, Naresh Sachdeva1,
Leonardo Davilla1, Gwendolyn B. Scott1, Charles Mitchell1,
Luis Cintron2, Jose Moreno1, Mobeen Rathore3, Isaac Delke3
1
University of Miami-Miller School of Medicine, Miami, Florida;
Borinquen Healthcare Center, Miami, Miami, Florida; 3University
of Florida, Jacksonville, Florida
2
Background and Objectives: Lymphocyte phenotyping is a standard
diagnostic procedure that provides valuable information for the diagnosis
and monitoring of patients with cellular immunodeficiencies such as
HIV/AIDS. During the past decade there has been a major advancement
in determination of lymphocyte subsets with the use of two colors to
multicolor flowcytometers along with the introduction of new softwares
for acquisition and analysis. We have completed a pilot study in our
laboratory to assess the influence of 4-color and 6-color flowcytometers,
and respective analytical software´s on the enumeration of lymphocytes
in HIV infected individuals. Methods: The expression of various cell
surface markers on lymphocytes were measured on the FACSCalibur
(4-color) and FACSCanto (6-color) flowcytometers from BD
Biosciences, San Jose, CA the software´s and reagents were supplied by
the same manufacturer – using the EDTA blood from 29 HIV infected
patients. The percentage of lymphocyte expressing a particular cell
surface marker of interest was calculated on the FACSCalibur using the
Cell Quest software; while the analysis on FACSCanto was done using
Cell Quest and FACS Diva software. The significance of difference
between the means was calculated by a paired t-test using the SPSS
software (version 13.0). Results: The data shows significantly higher
mean CD3 percentages on FACSCalibur (72.33±7.35) as compared to
FACSCanto (71.34±7.50) flowcytometer (p<0.05). When FACS Diva
software was employed on FACSCanto there was a further increase in
the mean CD3 percentages (73.59±7.41) (p<0.05). The CD4 cell
percentage seemed to be unaltered on FACSCalibur (28.53±12.21) and
FACSCanto (28.54±12.22). However; the CD4 cell percentage again
showed a significant increase with the use of FACSDiva software
(29.46±12.61) (p<0.05). The cytotoxic T-cells (CD8) and B-cell
percentages were unaffected when either of the instruments or software´s
was used. However; the NK cells did show significant differences in
their mean percentages. Overall, the means of lymphosum were
96.97±2.25 and 97.94±1.97 on FACSCalibur (with Cell Quest) and
FACSCanto (with FACSDiva) respectively with a significant difference
(p<0.05). Conclusions: Results of lymphocyte phenotyping affect
diagnosis, monitoring, therapy and prognosis in various infections such
as HIV. The differences in type of flowcytometers and softwares
influence the enumeration of lymphocytes subsets in particular, CD3,
CD4 and NK cells. It will be appropriate to suggest the laboratories
performing lymphocyte phenotyping should also report instrument and
software used for the specimen analysis.
255/P109
NOVEL CALIBRATION METHOD FOR FLOW
CYTOMETRIC FLUORESCENCE RESONANCE ENERGY
TRANSFER MEASUREMENTS BETWEEN VISIBLE
FLUORESCENT PROTEINS
Peter Nagy1, László Bene1, Manuela Braun2, Christof Antz2,
Jacques Paysan3, Gyorgy Vereb1, János Szöllõsi1
1
Biology, Debrecen, , Hungary; 2Otogene GmbH, Tübingen,
Germany; 3Darmstadt University of Technology, Developmental
Biology and Neurogenetics, Institute of Zoology, Darmstadt,
Germany
Fluorescence resonance energy transfer (FRET) is a sensitive method
for measurement of the interaction of biologically relevant molecules.
With the advent and wide-spread application of green fluorescent protein
(GFP) and its spectral variants the detection of protein associations in
living cells has also become possible. Accurate calibration of the FRET
signal is crucial for relible and reproducible measurements. In order to
quantitate the FRET signal we have used chimeras in which cyan
fluorescent protein (CFP) was separated by amino acid linkers of different
160
sizes from yellow fluorescent protein (YFP) and used them to calibrate
the cell-by-cell flow cytometric FRET measurements. We measured
donor, direct, and sensitized acceptor fluorescence intensities and
developed a novel way to calculate a spectroscopic constant that
characterized the fluorescence intensity of acceptor molecules relative
to the same number of excited donor molecules, which is essential for
quantifying FRET efficiency. This was achieved by calculating FRET
efficiency in two different ways and minimizing the squared difference
between the two results. Our method reliably detected the association of
Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency
between Cdk2 and a random peptide was negligible. We identified and
sorted subpopulations of yeast cells showing interaction between the
studied proteins. The calibration method we developed is
straightforward, easy to use and can accurately quantitate FRET
efficiency between GFP derivatives in flow cytometry.
256/P110
QUANTITATIVE AND STATISTICAL COMPARISON OF
UNIVARIATE FLOW CYTOMETRY DATASTETS USING
HISTOGRAM SIMILARITY MEASURES
Tytus Bernas1, Elikplimi Kwaku Asem2, J. Paul Robinson3,
Bartlomiej Rajwa4
1
Indiana; 2Indiana University, Pharmacology, School of Medicine,
& Biomedical Engineering, West Lafayette, Indiana; 4Purdue
Lafayette, Indiana
Comparison of fluorescence intensity distributions is an important
analysis step in many applications of flow cytometry. Since the
distributions (histograms) may have arbitrary shape, nonparametric tests
are used for such purpose. Nonetheless, classical tests, based on
Kolmogorov-Smirnoff or 2 statistics tend to overestimate probability
of uniqueness of compared flow cytometry histograms. Several methods
have been developed to alleviate this problem. However, none of these
techniques simultaneously provides a mathematical metric for histogram
comparison and statistics for determination of probability of histogram
uniqueness. Here we apply histogram similarity measures (EMD, QF
and HI, developed for image classification) to quantitatively compute
such distances in flow cytometry intensity distributions. The proposed
metrics do not require histogram binning or modeling of intensity
distributions. We use Monte-Carlo methods to obtain intensity histograms
derived from the same population of cells measured in a flow cytometer.
Furthermore, we incorporate cytometer calibration data into the
validation procedure to account for histogram variability caused by
instrumental factors. We construct distribution of histogram distances as
a function of the number of detected events (cells) and instrumental
noise. Using these data we estimate the probability that two histograms
separated by a given distance are statistically different. Finally we
demonstrate applicability of proposed metric in practical flow cytometry
area in the research and clinical laboratory.
257/P111
WHEN THE LYMPHOSUM DOES NOT ADD UP
Michèle Bergeron1, Tao Ding1, Nadia Soucy1, Naomi Lobo1,
Francis Mandy1
1
Public Health Agency of Canada, National HIV Immunology
Laboratory, Ottawa, Ontario, Canada
INTRODUCTION: Currently, most immunophenotyping guidelines
for T-cell subset determination of adults living with HIV recommend
the use of a single tube, four-color antibody panel (CD45/CD3/CD4/
CD8). However, the inclusion of B cells and Natural Killer (NK) cell
markers are beneficial as they provide additional internal quality control.
Traditionally, LymphoSum, the sum of T, B and NK cell percentages,
has provided an internal assessment of lymphocyte recovery.
Theoretically, the sum of the three cell types equals to 100% in a gate
without monocyte contamination. However, there are cases where the
LymphoSum is 3 to 5 % below the 100% mark. In such cases, the NK
cell values may be underestimated because they express surface antigens
at a lower level of fluorescence intensity. OBJECTIVE: The aim of this
study is to select a gating strategy to accurately assess NK cells with low
mean fluorescence intensity within the LymphoSum gate. It is to improve
the reliability of LymphoSum as an internal quality control check.
METHOD:
Fourteen (11HIV + and 3 HIV-) specimens were
immunophenotyped with the following two-tube four-color panels:
CD45/CD3/CD4/CD8 and CD45/CD3/CD16-56/CD19. The stained
preparations were lysed with ImmunoPrep reagent. An additional 0.5
ml of formaldehyde 2% was added. Absolute counts were calculated
with Flow-Count microfluorospheres. The analysis was performed on
a FacsCalibur. NK cell percentages (CD3-16+56+) were measured with
conventional gating (CD45+++ lymphocytes) and with a new “Accurate
NK” gating or ANK (CD45+++ not CD19+). Using the number of NK
events and lymphocyte events, a new NK percentage was calculated.
Both LymphoSums were compared. The ratio of median fluorescence
intensity of the NK positive over NK negative cell clusters was measured
and tested to see if it works as a gating criterion. RESULTS: The range
of the median fluorescence intensity of NK cell expression was between
39 and 149 relative linear channels (RLC) with NK+/NK- ratios between
5 and 25. Four specimens with ratios of 5 and 6 correlated with
LymphoSum < 97%. Following ANK gating, the LymphoSums of these
specimens were up to 99% with a 2 to 3 % NK cell increase.
CONCLUSION: The ANK gating improves the reliability of
LymphoSum, a useful internal quality control tool. This gating strategy
permits the accurate reporting of NK cell numbers. The ANK gating
strategy could be valuable for monitoring natural killer cell functional
studies where the variable levels of NK cell surface antigen expression
are of interest.
258/P112
EVALUATION OF MOUSE BONE MARROW BY FLOW
CYTOMETRY AFTER IN VIVO DOSING WITH
ERYTHROPOIETIN, GRANULOCYTE-COLONY
STIMULATING FACTOR OR 5-FLUOROURACIL
Cindy X. Zhang1, David McFarland2, Melanie Quinlan2, Jie
Ding2, Terry Sellers2, Daniela Ennulat2, Padma Kumar
Narayanan2, Heath Thomas2
1
Bristol-Myers Squibb Pharmaceutical Research Institute,
Pennington, New Jersey; 2GlaxoSmithKline, King of Prussia,
Pennsylvania
In pre-clinical toxicology studies, rodent bone marrow differentials are
routinely assessed by manual microscopic evaluation. This method can
be tedious, labor intensive and subjective with several hundred cells
being counted per slide. Application of flow cytometry to evaluate
mouse bone marrow subsets has been investigated to increase efficiency
and reduce statistical variance of bone marrow differentials. Human
recombinant erythropoietin (hrEpo), human granulocyte-colony
stimulating factor (hG-CSF), or 5-fluorouracil (5-FU) was used as tool
compounds to induce bone marrow changes. Male mice (3 groups of 5)
were given a single dose of hrEpo (0, 1000, 3000 U/kg) subcutaneously,
multiple doses of hG-CSF (0, 25, 50 µg/kg/day) subcutaneously, or
single dose of 5-FU (0, 50, 150 mg/kg) intravenously. Mouse bone
marrow cells were collected from both femurs 48 hours, 24 hours, or 5
days after the final dose, respectively. Cells from right femur were used
for marrow smears, and cells from left femur were flushed and prepared
for flow cytometry. Smears were stained and analyzed manually with
standard techniques. Myeloid, lymphoid, and nucleated erythroid cells
were enumerated, and myeloid : erythroid (M: E) ratio was calculated.
Cell suspensions for flow cytometric analysis were stained with antiCD45-FITC, CD11b-APC, Ter119-PE antibodies, Hoechst 33342 (HO)
and 7-actinomycin D (7-AAD). Cells were then washed and fixed
overnight in 1% paraformaldehyde. Flow cytometric analysis was
performed on a BD LSRII. Myeloid, lymphoid and nucleated erythroid
cells were defined as CD45 +/CD11b +/ HO+, CD45 +/CD11b-/ HO + and
CD45-/ter119+/HO+, respectively. 7AAD was used to exclude cells with
compromised plasma membranes. Results demonstrated that flow
cytometric analysis was sensitive to bone marrow perturbations induced
by tool compounds. Statistic analysis shows that there is a very good
correlation between flow cytometric and manual methods (r = 0.99),
although the absolute count of nucleated erythroid was consistently
lower when measured by flow cytometry. Further more, flow cytometry
produces less statistical variation in cell counts and is therefore a more
sensitive assay for detecting changes in the bone marrow compartment.
259/P113
PACIFIC ORANGE™ DYE FOR USE IN
POLYCHROMATIC FLOW CYTOMETRY EXPERIMENTS
USING VIOLET DIODE LASER EXCITATION
Gayle Marie Buller1, Stephen Yue2, Jixiang Liu2, William L.
Godfrey3
1
Molecular Probes, Inc., Flow Cytometry, Eugene, Oregon;
Molecular Probes, Inc, Chemistry, Eugene, Oregon; 3Molecular
Probes, Inc., Eugene, Oregon
2
The use of violet-excited fluorochromes in polychromatic flow
cytometry has been limited because most of these fluorochromes are
only bright enough for use on densely expressed antigens and are
sometimes significantly excited by a 488 nm Argon ion laser. With the
increasing prevalence of violet lasers on flow cytometers, there is a
demand for bright violet excited fluorochromes. We have developed a
novel violet-excited organic dye, Pacific Orange TM dye, which has an
emission maximum at 551 nm. The development of this dye addresses
the need to transfer well-resolved markers off of the 488 nm excitation
line and onto the violet laser, thus enabling the detection of other markers
with the 488 nm laser. Pacific OrangeTM dye is at least twice as bright as
the other available green-emitting violet-excitable dyes, Cascade
YellowTMdye and Alexa Fluor®430 dye, when read with a 530 nm band
pass filter, and can be even better resolved with a 575 nm bandpass
filter. Pacific Orange TM dye can be used for two color
immunophenotyping with Pacific Blue TM dye (450/50 nm band pass
filter) with minimal compensation and without 488 nm excitation. Data
is shown for human blood stained with anti-CD8 complexed with
Zenon® Pacific Orange TM mouse IgG1 labeling reagent and mouse antihuman CD4Pacific BlueTM. Pacific OrangeTM dye can easily be used for
CD45/SSC gating to better define a leukocyte gate and help in eliminating
unwanted debris. Pacific Orange TM dye can be used with the far red
emitting Quantum Dots ® 605, 655, and 705 to perform a 5-color
immunophenotyping with only violet laser excitation. Finally, Pacific
OrangeTM dye can be paired with Pacific BlueTM dye and a fixable dye
with emission maximum around 515 nm to exclude dead cells from a 2color immunofluorescence stain using only violet excitation.
260/P114
MULTICOLOUR FLOW CYTOMETRY TO QUANTIFY
INTERNALIZATION OF MONOCLONAL ANTIBODIES. A
NOVEL METHOD CONFIRMED BY CONFOCAL
MICROSCOPY
Amir H. Iranpour Feridani1, Bo Baldetorp1
1
Lund University, Lund, Sweden
Background: Some therapeutic monoclonal antibodies (Mab), like
BR96, have the unique ability to internalise when bound to its antigen
on the cell surface. If these are labelled with radionuclides or toxins, the
whole immunoconjugate can be incorporated within the cells and cause
an effective cell destruction. When BR96 is bound to its antigen Lewis
Y, the complex will actively internalize into the cell. Flowcytometry
(FCM) enables quantitative measurements on thousands of individual
cells. FCM together with qualitative analysis confocal microscopy (CM)
was used for analysis of internalization. Material and Methods: A rat
colon-tumour cell line H1D2-WT, expressing Lewis Y antigen was used.
BR96 was conjugated with fluorescein isothiocyanate (FITC).
Corresponding anti-IgG antibody was conjugated with phycoerythin
(PE) and used for detection of the non-internalized antibodies at the cell
surface. Quantitative FCM analysis was performed after 0, 1, 2, and 4
hours of incubation with the FITC conjugated mAbs and subsequent
binding of anti-IgG mAb to all cells. Proportion of antibody, which was
equivalent to the grade of internalization, remaining at the cell surface
at various time points was analyzed as the ratio of FITC/PE. Results:
Quantitative measurements with FCM indicate that a proportion of antiBR96 antibodies remaining at the cell surface, declined after 1-2h. This
is consistent with the findings from the qualitative CM, where after 1h,
a clear internalization could be detected. Cells stained with TUNEL
texas Red detected with CM, indicate that BR96 by itself is toxic to the
cells, where the nuclei of the cells becomes damaged. Conclusion: Present
data suggest that our novel quantitative multicolour FCM method
supported by CM is useful for measuring the grade of antibody
internalization. Furthermore, FCM data indicate the existence of
subpopulations with different degrees of internalization, that is verified
with CM. CM also shows that BR96 has toxic effect by itself.
161
261/P115
INTRODUCTION OF 7-LASER LSRII AND 5-LASER
FACSARIA
David Dombkowski1, Larry Duckett2, David Matsuyama2,
Stephen Ziganti2, Frederic Preffer1
1
Center for Regenerative Medicine, Massachusetts General Hospital,
Boston, Massachusetts; 2BD Biosciences, San Jose, California
263/P117
DEVELOPMENT OF MICROFLUIDIC STRUCTURES FOR
HIGH THROUGPUT FLOW CYTOMETRIC
CHARACTERIZATION OF BLOOD CELLS
Jörg Neukammer1, Janko Theisen2, Kerstin Brattke1, Andreas
Kummrow1, Thilo Guschauski2, Martin Schmidt2
1
Physikalisch-Technische Bundesanstalt, Berlin, Germany;
Technical University Berlin, Institute for Engineering Design,
Micro and Medical Technology, Berlin, Germany
2
The advent of low power solid state air-cooled lasers has been associated
with a tremendous improvement in flow cytometric technology, typified
by the release of the LSR-2 in 2002 and the BD-FACSAria in 2003.
When properly combined into flow cytometers configured with
correlated improvements in optical and electronic design, these
physically smaller, more economically operated fixed-line lasers provide
excellent results. Unlike water cooled ion-gas lasers which can be
variably tuned to various useful output lines, these newer air-cooled
lasers are relatively small and competitively priced low enough to
conceptually just add another laser, should an excitation line be deemed
necessary to adapt to changing research demands. The appropriate
integrated use of multiple excitation sources with suitable fluorochromes
should permit reduced spillover correction/compensations between all
fluorescent parameters, in contrast to the relatively higher levels we
currently must sometimes apply [~20-40%] with the FACS Vantage SE/
DiVa. Therefore, the aim is to use more excitation lines, with fewer
fluorochrome excitations per line. The new design goals are improved
measurements (photon counting statistics) by combining increased laser
powers (e.g., up to 100 mW @ 488 nm,) and multiple laser excitation
line flexibility with enhanced optical collection efficiency to minimize
measurement error and improve resolution of relevant biological
populations. Associated improvements in cytometer optical design
include quartz-cuvette laser interrogation [in Aria], directly coupled
independent fiber-optic routing of emission signals for up to 7 laser
excitation lines [in LSR] and octagonal photomultiplier configuration.
The high performance, compact air-launched designs to route selected
excitation lines and spatially separated emission signals no longer restricts
lasers and photomultipliers to orthogonal planes, relative to the sample
stream. This allows this instrument design to be constructed with a
significantly smaller footprint than previous cytometers. The Special
Order Arias and LSRII systems configured to the researchers needs
exemplify these designs and concepts to provide the tools required to
pioneer inroads in flow cytometric discoveries. In this poster, we will
present our first experience with this new line of instrumentation.
262/P116
FLOW CYTOMETRIC DETECTION OF ERYTHROCYTE
ZINC PROTOPORPHYRIN IN IRON DEFICIENT
PATIENTS
Jörg Neukammer1, Benedikt Krämer2, Andreas Kummrow1,
Sandra Schädel1, Silke Heller3, C. Thomas Nebe4
1
Physikalisch-Technische Bundesanstalt, Berlin, , Germany;
PicoQuant, Berlin, Germany; 3Sankt-Gertrauden-Krankenhaus,
Zentrallaboratorium, Berlin, Germany; 4Klinikum Mannheim,
Zentrallabor Inst. Klin. Chemie, Mannheim, Baden-Württemberg,
Germany
2
Iron deficiency leads to an increased Zinc protoporphyrin (ZnPP)
concentration in red blood cells. To detect individual erythrocytes
exhibiting increased ZnPP content by flow cytometry, we employed
krypton ion laser radiation with a wavelength of 413.1 nm. Fluorescence
of ZnPP was observed using a band filter centered at 625 nm, the full
width at half maximum amounted to 30 nm. With increasing (average)
ZnPP concentration the histograms, representing the distribution of the
fluorescence of erythrocytes, were observed to become more
asymmetric. The number of erythrocytes exceeding a specific
fluorescence intensity in the ZnPP emission channel was determined
and compared to results obtained by standard fluorometry of washed
red blood cells and by serum ferritin measurements. Flow cytometric
detection of ZnPP containing erythrocytes is expected to be more sensitive
against acute changes compared to the conventional fluorometric
detection of ZnPP in the total (washed) erythrocyte population.
162
Microdevices integrating all elements required for a dedicated flow
cytometric diagnostics are highly interesting since they could be designed
for one way use in a cost-efficient production line. In addition,
microstructures allow to combine measuring quantities not easily available
in conventional flow cytometry. We have designed different
microstructures for optical and impedance analysis of single particles.
LIGA technique (German acronym for lithography, electroplating, and
molding) and ultraprecision milling was deployed to fabricate the mold
inserts. The latter technique is recommended for convenient
implementation of three dimensional fluidic structures, required for
two-dimensional hydrodynamic focusing. Hot embossing served to
obtain polymethylmethacrylate or polycarbonate microstructures.
Subsequently, upper and lower parts were assembled using epoxy resin
or by laser welding. We will present first results of optical extinction and
light scattering obtained from microstructures with an integrated
monomode fiber, multimode optical fibers as well as electrodes for
impedance measurements. The monomode fiber was used to guide laser
radiation at 488 nm and 633 nm to the interaction region, the multimode
fibers were arranged to measure the extinction signal and to collect light
at different angles. Fluorescence, emitted perpendicular to the direction
of particle flow and to the k vector of the exciting laser beam, was
detected through a long working distance microscopy objective (40x,
0.5).
264/P118
GATING METHODS FOR FLOWCYTOMETRY IN MULTIDIMENSION SPACE
Danhua Zhao1
1
Gating plays an extremely important role in flowcytometry. The number
of channels has been increasing over the last several years to enhance
the performance of the flowcytometer. This paper, however, proves
that such an improvement is also affected and even limited by the
correlation between channels. In other words, any additional channel
which has high correlation with other channels does not significantly
improve the separation of populations. One challenge arising from
such an increase is the difficulty of gating individual populations.
Solutions have been sought to address this problem. One possible solution
is to take a non-graphic linear algebra approach. Advantages of this
approach include (1) removal of display limitation; (2) ease of
automation; (3) less dependency on dimensionality.
265/P119
GEOMETRIC ASPECTS OF CELL MEMBRANE
MEASUREMENTS AND INSTRUMENT DESIGN
Gordon W. Wiegand1
1
G.W Consultancy, Havre de Grace, Maryland
Introduction Flow and image cytometers are often used in challenging
ways in the field of virology. While much attention has been applied to
multi-color immunophenotyping, other techniques present unique
challenges. Proteins originating from genes such as TSG 101, etc are
suspected of being involved in viral trafficking and cell infectivity.
Protein copy number is typically low, therefore, fluorescent antibodies
raised against these molecules produce very low intensity signal. My
primary objective was to image Influenza infected MDCK cells to
determine where specific proteins are located within the cell membrane
and /or endo-membrane region of the cytoplasm. Secondly, I have been
working to enhance our cell sorter´s fluorescence sensitivity to a level
that is comparable to critical microscopy. I approached both of these
objectives from a geometric prospective for cell manipulation and
detection engineering. Sub-micron and nanometer prospective of cell
membrane measurement. I used Scanning Confocal Microscopy to
associate lipid raft laterally located within the membrane to tsg101
observed in the outer cytoplasm. Muti-color images were displayed in 2
and 3 dimensions after exposure to a high resolution CCD array. I used
Transmission Electron microscopy to detect tsg101 on a molecular
level and associate it with viral budding within the cell membrane. The
density of tsg101 within a nano-arch of the membrane was determined
by assuming the cell membrane width to be 5 nm thick then counting
the silver enhanced anti-tsg101 within a section of the arch. This value
was then geometrically converted to tsg101 molecules / cell surface.
Ultimately, the need is to sort cells selected through invitro knockout
and antibody resistance by flow cytometry. Therefore, I designed and
constructed a flow cytometry test bench used to evaluate optics and
other systems critical to enhancing the sensitivity of our FACSVantage.
I placed greatest emphasis upon the geometry of various fluorescence
lenses working distances and system light gathering efficiency.
266/P120
RESOLUTION REQUIREMENTS FOR THE
DIGITIZATION OF FLOW CYTOMETRY DATA
James C. S. Wood1
1
Consultant, Galax, Virginia
Historically, wide dynamic range data has been presented in 4-decade
1024 channel logarithmically scaled histograms. The data was scaled
with an analog logarithmic amplifier and digitized with an analog to
digital converter (ADC) whose output was truncated to a resolution of
10 bits. In order to achieve the same resolution of 256 channels in the
first decade of a 4-decade logarithmically scaled histogram by directly
digitizing the linear electronic signal from the photodetector, the
electronic signal must be digitized with an ADC that has at least 21 bits
of dynamic range. This is available commercially for instruments that
digitize the pulse height and/or pulse area. However, instruments that
digitize the data pulse and use the digitized waveform to calculate the
pulse height and pulse area use oversampling to achieve dynamic ranges
approximately an order or magnitude lower than the 21 bit dynamic
range. The more limited resolution of the pulse digitizing instruments
is a result of the lack of availability of economical fast ADC’s with
sufficiently high enough resolution.
The question remains unanswered
as to what impact the resolution/dynamic range of the digitization has
on the subsequent analysis of flow cytometry data. To answer this
question, mathematical models have been made to explore how the
requirements for resolution/dynamic range of the data digitization are
dictated by the required precision of the measurement. The analysis of
populations with larger coefficient of variations does not need high
resolution digitization. This is most noticeable for populations in the
first decade of a 4-decade histogram. Additionally, the limitations of
the use of oversampling to increase the resolution are investigated to
determine under what conditions is it appropriate and how much
oversampling is required to boost the dynamic range of a lower resolution
ADC to the required higher dynamic range.
267/P121
A 16-CHANNEL AVALANCHE PHOTODIODE DETECTOR
ARRAY FOR VISIBLE AND NEAR-INFRARED FLOW
CYTOMETRY
William G. Lawrence1, Christopher Stapels1, Richard Farrell1,
Joseph Tario2, Edward Podniesinski2, Paul Wallace2, James
Christian1
1
2
Radiation Monitoring Devices, Watertown, Massachusetts;
Roswell Park Cancer Institute, Buffalo, New York
We report on the development and application of a flow cytometer
using a 16-channel avalanche photodiode (APD) linear detector array.
The array is configured with a dispersive grating to simultaneously
record emission over a broad wavelength range using the 16 APD
channels of the linear array. The avalanche photodiode detector elements
have a peak quantum efficiency of 80% near 900 nm and have at least
40% quantum efficiency over the 400-nm to 1000-nm wavelength
range. The wide wavelength sensitivity of the APD array permits the
use of multiple excitation sources and many different fluorescent labels
to maximize the number of independent parameters in a given
experiment. The extended red sensitivity of the detector array facilitates
the use of lower energy excitation sources and near IR emitting dyes
which reduces the impact of autofluorescence in signal starved
measurements. We show the sensitivity and linearity measurements for
a single APD detector. Initial results for the flow cytometer with the 16element APD array and the 16-channel readout ASIC (application specific
integrated circuit) are presented.
268/122
USE OF VIOLET-EMITTING AMINE REACTIVE DYE
COMPARED TO ETHIDIUM MONOAZIDE (EMA) FOR
LIVE-DEAD DETERMINATION IN INTRACELLULAR
STAINING ASSAYS
Martin Bigos1, Ck Poon2, Elizabeth Sinclair3
1
Gladstone Institute of Virology and Immunology, San Francisco,
California; 2University of California, San Francisco, GCRC Core
Immunology Laboratory, San Francisco, California; 3University of
California, San Francisco, California
Intracellular staining assays are commonly used in flow cytometry to
measure immune function of different cell subsets. These involve a
significant period of incubation with stimulants, and may be performed
on frozen specimens from dubious storage conditions. For these reasons
the use of a live-dead cell marker is usually necessary to get reproducible
quality data. Because of the need to permeablize the cells for staining,
traditional intercalating DNA reagents such as PI or DAPI are not usable.
EMA can be photoactivated to covalently bond to DNA has been the
only option available for this situation. It is disadvantageous because a)
it requires an extra step (the photoactivation), b) results are variable
depending upon the light source, c) it has significant spectral overlap
with a large number of fluorochromes commonly used for phenotyping.
Molecular Probes (Invitrogen) has released a number of amine reactive
dyes with differing spectral properties. These dyes specifically bind to
amines intracellularly and on the surface. Since the amount bound in
these two compartments is markedly different, it is easy to discriminate
between live and dead cells. Moreover, excess dye can be washed away,
preserving the discrimination post-permeabilization. This poster
compares the staining properties of EMA and the violet emitting amine
reactive dye (VARD). We show that they stain the same cells, and compare
their effects spectrally in an intracellular staining assay and compare the
results.
269/P123
VALIDATION OF POLYCHROMATIC STAINING PANELS
TO DETECT T CELL SUBSET CYTOKINE RESPONSES
ON THREE FLOW CYTOMETER PLATFORMS
Bridget E. McLaughlin1, Nicole Baumgarth2, Martin Bigos3,
Stephen C. De Rosa4, John D. Altman5, Mario Roederer6,
Douglas Nixon3, Janet Ottinger7, Judy Li8, Laurel A
Beckett8, David M Asmuth1
1
University of California, Davis, Internal Medicine, Division of
Infectious Diseases, School of Medicine, Davis, California;
2
University of California Davis, Center for Comparative Medicine,
School of Medicine, Davis, California; 3Gladstone Institute of
Virology and Immunology, San Francisco, California; 4University of
Washington, Fred Hutchinson Cancer Research Center, School of
Medicine, Seattle, Washington; 5Emory University, Emory Vaccine
Center at Yerkes, School of Medicine, Atlanta, Georgia; 6National
Institutes of Health (NIH), NIAID, Vaccine Research Center,
Bethesda, Maryland; 7Duke University Medical Center, Duke
Center for AIDS Research, Duke Center for AIDS Research,
Durham, North Carolina; 8University of California Davis, Division
of Biostatistics, School of Medicine, Davis, California
Background: Polychromatic flow cytometry allows simultaneous analysis
of lymphocyte maturation marker surface expression and intracellular
cytokine expression to identify rare populations of antigen-specific
cytokine secreting T cells. While efforts to standardize intra-laboratory
assays have been published, we sought to investigate the bias and
variability of a 9-color panel on three different cytometer platforms.
Methods: An empiric stepwise approach was taken, testing 30
permutations of 6-color surface marker reagent combinations using
PBMCs from a single donor. The 2 best panels were expanded to include
antibodies specific for three intracellular cytokines and were repeatedly
tested (on 3 separate occasions) using PBMCs (cryopreserved at a single
time point) from 3 donors on 3 flow cytometers (BD Aria, LSR II, and
Dako-Cytomation MoFlo). Naïve cells (N) are defined as CCR7 + /
163
CD45RA+; Central Memory (CM) as CCR7+/CD45RA-; Effector Memory
(EM) as CCR7-/CD45RA-; and RA + Memory (RAM) as CCR7-/
CD45RA+.Results: Panel selection: Two 9-color panels that exhibited
robust T-lymphocyte subset enumeration and minimal spillover in
channels used for cytokine detection were identified (Table I). Phenotype/
maturation subsets: Three-way (donor-panel-instrument) and two way
interactions were examined and no systematic differences were detected
between individuals across all 3 instruments regardless of reagent panel.
Thus, ANOVA main effects models of SEB stimulated samples revealed
only minimal differences in selected subsets between instruments (CD4
N and CD4 RAM [p=.003 and <.001, respectively]) and between reagent
panels (CD4 CM and CD4 N [p<.001 and p=.04, respectively]). No
differences were seen for CD8 populations. Cytokine expression:
Similarly, there was little evidence for differences between instruments
in measurement of cytokine expression, with significant F statistic values
in isolated examples, notably the fraction of IFN-ã positive CD4 CM
and EM subsets. In contrast, differences were observed in cytokine
frequency between the reagent panels, more often in the CD8 than CD4
subsets. These differences will be discussed.Conclusions: Cross instrument
results are comparable though optical configurations influence the
sensitivities in certain channels. Variability and sensitivity in the
measurement of lymphocyte subsets and cytokine expression across
multiple cytometer platforms can be predicted and used to design clinical
trial endpoints.
apoptosis, cytokine production and protein transfection. Flow Cytometry
Fluorescence Intensity measurements are often quantitated with beads
calibrated in Molecules of Equivalent Soluble Fluorochorome (MESF).
In addition to Fluorescent Intensity measurements, the Cell Lab Quanta™
Series Flow cytometers provide the ability to simultaneously measure
the Cellular Volume using the Coulter principle. This unique
combination of parameters provides the opportunity to calculate either
Fluorescence Concentration (FC) for intracellular stains, or Fluorescence
Surface Density (FSD) for surface markers. Fluorescence Concentration
(FC) is defined as the amount of fluorescence per cellular volume (u3).
FC is applicable to dyes, probes and fluorochromes targeted to
intracellular structures like; nuclear DNA, RNA, mitochondria, nuclear
and cytoplasm proteins, intracytoplasm antigen-antibody reactions and
the expression of fluorescent proteins such as GFP, YFP, CFP, DsRED.
Fluorescence Surface Density (FSD) is defined as the amount of
fluorescence per unit of area (u2). The main application of FSD is to
measure the concentration of active sites on a cell using fluorescent
surface markers ie specific monoclonal antibody or other conjugate.
Data showing the application of the FC parameter in the analysis of the
expression of GFP in a generic cell line after transfection, and the FSD
parameter in the analysis of Annexin V-PE positive apoptotitc cells is
presented. GFP and PE fluorescence were calibrated with beads with
known Molecules of Equivalent Soluble Fluorochorome (MESF). NIST
traceable beads were use to calibrate the Mean Cell Volume. Other
examples performed with different size fluorescent beads illustrate the
use of FC and FSD. The addition of these two unique quantative
parameters make the Cell Lab Quanta™ Series Flow Cytometers a
powerful tool in cellular research.
272/P126
INDO-US CYTOMETRY WORKSHOPS
Awtar Krishan1, L.Scott Cram2
1
University of Miami, Pathology, Miller School of Medicine,
Miami, Florida; 2Los Alamos National Laboratory, Los Alamos,
New Mexico
270/P124
OPTICAL FILTERS IN PRACTICE
Gouzel Tokmoulina1
1
HHMI at Yale University School of Medicine, Cell Sorter Core
Facility, School of Medicine, New Haven, Connecticut
Optical filter configuration is crucial to the operation of any flow
cytometer. While straightforward in theory (with all the reference material
widely available in flow cytometry books and on the web), the task of
selecting the right optical filter is more difficult in practice. We discuss
our experience with a performance of two dichroic filters for He-Ne
633 nm excitation path in detection of APC, Alexa 680 and APC-CY7
fluorochromes. Initial testing of the complete configuration resulted in
a low fluorescence signal even for the brightest channel. The reflectance
curve for the first dichroic filter later provided by the manufacturer
showed efficiency as low as 60-65%. Combined with reflection from
the second dichroic, the intensity delivered to one of the detectors
might be as low as 36%. As a result, we conclude that both the
transmission and the reflectance characteristics for dichroic filters must
be considered when choosing an optimal set. Furthermore, the overall
effect of all involved filters must be taken into consideration for the
highest fluorochrome intensity.
271/P125
QUANTITATION OF FLUORESCENCE
CONCENTRATION AND FLUORESCENCE SURFACE
DENSITY USING THE BECKMAN COULTER® CELL LAB
QUANTA™ SERIES FLOW CYTOMETERS
Michael Thomas1, Raquel Cabana1, Richard Thomas1
1
NPE Systems, Inc., Pembroke Pines, Florida
Quantitation of fluorescence in Flow Cytometry has become increasingly
important. In certain diseases a relationship has been drawn between the
index of activity or effectiveness of a cellular process and the amount of
fluorescence. Examples of this are: cellular proliferation, differentiation,
164
The Indo-US Cytometry workshops seek to interface experts in
cytometry with researchers in India and neighboring countries who
desire training in the latest analytical methods. Lectures by experts from
USA, UK, Canada, Europe and India are followed by wet lab sessions
and tutorials. The First workshop in 2001 was held at the Punjab
University in Chandigarh. Thirty- five students and ten faculty members
participated in this weeklong workshop. The 2nd workshop on the
“Applications of Flow Cytometry in Molecular and Cellular Biology”
was hosted by the Centre for Cellular and Molecular Biology in
Hyderabad. Twenty-seven participants, fifteen faculty members attended
this workshop. The 3rd Workshop on “Applications of Flow cytometry
in Drug Mechanistics” was hosted by the Regional Research Laboratories,
CSIR, Jammu, in September 2003. The 4th Indo-US Cytometry
Workshop was hosted by the Advanced Center for Treatment, Research
and Education in Cancer, Tata Cancer Center, in Bombay. Twentyseven researchers, twelve faculty members from overseas and ten faculty
members from India gave lectures and supervised the wet labs. The 5th
workshop on “Applications of Cytometry in Malignant, Infectious and
Parasitic diseases” was hosted by the Panjab University and Post-Graduate
Medical Institute in Chandigarh. Thirty-five students and 20 faculty
members participated. The 6th workshop on “Monitoring of Stem cell
Phenotype” is hosted by SCT Institute for Medical Sciences and
Technology in Trivandrum from February 5-11, 2006. Thirty students
and 27 faculty are expected to participate in this workshop. In addition
to instruments at the host institutions, Becton Dickinson India Pvt. Ltd,
Beckman Coulter International SA, NPE Systems Inc, Guava Technology
Inc. and DakoCytomation have brought their latest instruments and
staff to the workshops. Support from ISAC, International Union Against
cancer (UICC) , NIH, Indo-US Technology Forum and other Indian
funding agencies has been critical for success of these workshops.
273/P127
FLOW CYTOMETRY COURSES FOR AFRICA
1
2
Claudio Vallan , Jennifer A. Wilshire , Adam Treister
3
1
University of Bern, Bern, Switzerland; 2Tree Star, Inc., New York,
New York; 3Tree Star, Inc., Ashland, Oregon
275/P129
IMMUNOFLUORESCENCE STANDARDIZATION TO
MEASURE THE NUMBER OF ANTIBODIES BOUND PER
CELL
Doug Redelman1
1
The UN Human Developing Index (HDI) provides a way to compare
the well-being of people in most of the countries worldwide. The statistic
divides 177 nations into states with high (57), medium (88) and low
(32) human development. All but two of the 32 countries exhibiting
low human development are located in Africa. Egypt and South Africa,
the two most developed African countries, are found at places 119 and
120. While the HDI is generally improving for most countries in the
world, in Sub-Saharan Africa it shows a steady decline. HIV/AIDS is
being seen as the principal cause for this. Human development in African
countries could hence be ameliorated by any contributions improving
the health care system. To this end any possible effort should be made
to benefit research within the African continent. Tree Star has developed
the FlowJo Africa program that gives away the FlowJo analysis software
for free to anyone conducting research in Africa. Within this initiative
we organized a one-week FlowJo tutorial in Bamako, Mali. In addition
this seminar covered general flow cytometry topics. The experience
disclosed that general flow cytometry courses probably would be helpful
and well accepted by the African scientific community. As of the
beginning of 2006, FlowJo Africa has distributed over 60 free software
licenses to 17 sites in Africa. This poster reports on our first training
session in Mali, and our plans to provide further resources to research
and clinical labs using flow cytometry in Africa.
274/P128
CALIBRATION OF QUANTUM DOTS AS PROBES OF
MOLECULAR ASSEMBLIES ON BEADS AND CELLS IN
FLOW CYTOMETRY AND MICROSCOPY
Yang Wu1, Samuel K Campos2, Gabriel P. Lopez3, Michelle A
Ozbun2, Larry A. Sklar1, Tione Buranda1
1
University of New Mexico, Pathology and Cancer Center, Health
Sciences Center, Albuquerque, New Mexico; 2University of New
Mexico, Molecular Genetics & Microbiology, Albuquerque, New
Mexico; 3University of New Mexico, Chemical and Nuclear
Engineering, Albuquerque, New Mexico
Background. Quantum dots are fluorescent semiconductor nanoparticles
with tunable optical properties. They have very high absorption crosssections and narrow emission bandwidths that make them highly suitable
for use as reporters for multiplexing assays on cells or beads. Quantum
dots are now widely used in cell based assays, however enabling methods
that allow for the quantitation of surface density of reporter quantum
dots are presently lacking in the literature. Methods. Here we describe
a simple calibration method that can be used to determine surface
coverage of quantum dot-tagged assemblies on beads or cells by flow
cytometry. We have compared the photophysical properties of,
commercially available, streptavidin-functionalized quantum dots
(Qdot525, Qdot585 and Qdot605) on beads and in solution and
compared them to fluorescein and fluorescein conjugates. Based on
their photophysical characteristics, we also assess the practical limits in
advantages of quantum dots over conventional fluorophores, in terms
of their relative absorption cross-sections and emission quantum yields.
Results. While the molar absorptivities of quantum dots are very high
over a very broad spectral range, the emission yield of quantum dots is
low relative to the fluorescein standard (e.g. 30% for Qdot525), but are
comparable to some fluorescein conjugates. Because of the relatively
narrow spectral widths of Qdot emission, most of the integrated emission
band falls within the bandwidth of conventional bandpass filters used in
flow cytometry and microscopy (e.g. >75% for Qdot525). In contrast,
for fluorescein, ≈ ≈ 47% of integrated emission intensity is transmitted
through a conventional 530 bandpass filter. The detection limits, and
surface coverage of quantum dots and fluorescein biotin were assessed
on beads in terms of numbers fluorophores/bead. Subsequently beads
of known surface coverage were used to characterize the surface
coverage of epidermal growth factor receptors (EGFR) on A431 cells
using biotinylated EGF ligand.
Conclusions. Characterization of the photophysical properties of
quantum dots in direct comparison to the long-standing industry
standard, fluorescein, in solution and on beads, presents a useful approach
to establishing calibrated uses of quantum dots in cell staining and other
bioanalytical measurements.
Sierra Cytometry, Reno, Nevada
In human clinical cytometry, there are at least two tests that measure the
quantitative expression of molecules per cell, namely, CD38 on CD8 T
cells and CD64 on monocytes. In order to make these measurements,
kits have been developed that contain reference materials and fluorescent
antibodies with known numbers of molecules of equivalent soluble
fluorochrome (MESF) per antibody. In addition, there are systems
available for quantitating IgG bound in indirect immunofluorescence
measurements, but these systems can be difficult to apply if multiple
antibodies are required to identify the cells of interest. In order to
overcome these limitations and devise more generally applicable
standardization procedures, strategies have been developed that utilize
commercial materials and/or locally prepared reagents.
The strategy
is based upon determining the specific fluorescence of a fluoresceinated
IgG (FITC-IgG) and then using this reference IgG along with anti-Ig
capture beads to determine the specific fluorescence of other IgG
antibodies labeled with fluorescein or other fluorochromes. Specific
fluorescence (not the same as the absorbance “F:P” ratios) can be
determined with a standard flow cytometer by measuring solutions with
known concentrations of fluorescein or of the reference FITC-IgG as
will be described. One could directly measure the specific fluorescence
of all the FITC-antibodies to be examined, but it requires much less
material to determine these values indirectly using anti-Ig capture beads.
Furthermore, capture beads can be used with antibodies labeled with
other fluorochromes that could be difficult if not impossible to measure
in the same way as fluorescein. To make the needed measurements, the
flow cytometer is calibrated with beads having known numbers of
fluorescein MESF (commercially available). The anti-Ig capture beads
are then loaded with the reference FITC-IgG and the Ig-binding
capacities determined based on the specific fluorescence. Since there
are commercial anti-Ig capture beads with stated Ig binding capacities
one might conclude that it is unnecessary to use the reference IgG.
However, the stated Ig-binding capacities may not be correct as will be
shown. Using these procedures, one can determine the specific
fluorescence of antibodies labeled with fluorescein, phycoerythrin (PE),
allophycocyanin (APC) or with virtually any fluorochrome including
PE or APC tandem conjugates for which standards are not available.
By preparing anti-rat or anti-hamster Ig capture beads and calibrating
rat or hamster FITC-IgG one can extend this strategy to include rat and
hamster monoclonals that are relevant to research studies of murine
cells. The result is to be able to enumerate antibodies bound per cell.
276/P130
INSTRUMENT CALIBRATION AND INTENSITY
MEASUREMENTS IN WIDE-FIELD AND CONFOCAL
FLUORESCENCE MICROSCOPY
Michael A. Model1, James L. Blank2
1
Kent State University, Biological Sciences, Arts & Sciences, Kent,
Ohio; 2Kent State University, Biological Sciences, Kent, Ohio
We propose two types of intensity standards for wide-field and confocal
fluorescence microscopy and describe their use. In many biomedical
applications, the aim of quantitative microscopy is to compare the
fluorescence intensity of specimens analyzed over a long time period,
possibly on different microscopes. This can be accomplished using a
standard with reproducible quantum yield. For other applications, the
absolute number of fluorescent molecules can be of interest. In this
case, a fluorescent intensity standard must carry a specified number of
fluorophores per unit area, and the excitation and emission spectra of
the standard must be the same as the dyes in the specimen.
A
standard of the first type can be based on a highly concentrated solution
of a dye, such as sodium fluorescein, Rose Bengal, Acid Fuchsin, or
Acid Blue 6. A standard slide is prepared by putting several microliters
of a concentrated dye under a cover glass. The critical feature of
concentrated dyes is strong light absorption at the excitation wavelength
leaving only a sub-micrometer outermost layer of liquid exposed to
light. This results in a number of desirable properties, such as stability
under illumination and good reproducibility. Concentrated dye standards
are easy to prepare, their emission is insensitive to the thickness of the
liquid between a cover glass and a slide, and their brightness is
165
comparable to the brightness of typical biological specimens. By using
common chemicals, dependence on a unique commercial product is
eliminated. Because the fluorescent field is uniform, concentrated
solutions of dyes can also be used to for shading correction. The main
downside of concentrated standards is that their fluorescence spectra
may be different from the spectra of the dyes in a specimen; in order to
relate data from different instruments, the optical filters should be kept
consistent.
A standard of the second type can be prepared from
fluorescently labeled erythrocytes. The average amount of label per
cell is first measured on a calibrated flow cytometer. For microscopy
observation, the cells are made to flatten on a glass slide so that their
thickness falls below optical resolution. By correlating the flow
cytometry data with the average gray level of flat cells measured under
a microscope, calibration of a microscope in the absolute units of
fluorophore per unit area can be achieved
in much the same fashion as building a flowchart. It provides both an
intuitive user interface and automated workflows that streamline data
acquisition, analysis, and visualization. The iGeneration workflow
defines both the data acquisition workflow as well as the data analysis
workflow. The highly flexible workflow architecture allows users to
customize and automate novel assays such as multi-scale tissue analysis.
Another promising application is the combination of quantitative laser
scan imaging and high-resolution confocal imaging, taking advantage
of both technologies within the same assay. We will demonstrate how
the flexible architecture and visual programming tools of the iGeneration
software can benefit assay development in different scenarios: • Using
pre-defined workflow with simple change of parameters for routine
assays • Creating custom workflows using standard algorithms and
features • Creating the custom modules to develop new algorithms and
features.
277/P131
A NEW SENSITIVITY TEST FOR BD FACSCANTO™
FLOW CYTOMETERS
279/P133
PROPOSAL OF STANDARDS FOR COMPARING THE
PERFORMANCE AND SUITABILITY OF MULTIPLE
CYTOMETRY TECHNOLOGIES
James E. Bishop1, Robert A. Hoffman1, Mahrukh A. Huseni1,
Hemangini Shah1
1
A quantitative test to measure sensitivity of fluorescence detectors of
BD FACSCanto flow cytometers was recently developed. The test is an
added functionality to existing cytometer setup and QC, using BD FACS™
7-color setup beads with BD FACSCanto clinical software, version 2.0.
The sensitivity value reported is a measure of the ability of each detector
to resolve dimly stained cells from negative cells. The sensitivity
measurement is a stain index (David Parks, Stanford University). For
calculation of sensitivity, fluorescence of the unlabeled bead (UNL)
and each detector´s primary fluorophore bead (POS) of BD FACS 7color setup beads are measured. Sensitivity is calculated as (normalized
MFIPOS – MFIUNL)/widthUNL, where MFI is median fluorescence intensity
and width UNL, is effectively 2 x standard deviation of the unlabeled
bead. The MFIPOS is normalized by a scaling factor that is proportional
to the MFI of lymphocytes stained with CD4 conjugates of each
fluorophore; thus, the sensitivity value of each detector reflects the
intrinsic brightness of the primary fluorophore measured therein. Use
of actual fluorophore-conjugated beads in BD FACS 7-color setup beads
(e.g., FITC beads, PE beads, etc.) made possible this normalization, and
consequently made possible establishment of absolute pass/fail values
for each detector applicable to any BD FACSCanto flow cytometer.
Studies showed that the sensitivity value for each detector includes
contributions from Q (fluorescence detection efficiency) and B
(background signal). Decrements in Q were induced by placing neutral
density filters in the laser beam. For filters to 10% transmittance,
sensitivity values, resolution of CD4-stained monocytes from negative
lymphocytes, and resolution of dimly stained populations in 6-color
stains were shown to decrease approximately in parallel. Increases in B
were induced by adding free fluorophore to both 7-color setup beads
and to cells stained with CD4-fluorophore. Increasing amounts of free
fluorophore caused approximately parallel decreases in sensitivity and
resolution of CD4-stained monocytes. In conclusion, the sensitivity
test, using BD FACS 7-color setup beads, was shown to quantitatively
measure the ability of each fluorescence detector to resolve dimly stained
populations. This sensitivity value reflects the intrinsic resolution
capability of each fluorophore, and effectively measures changes in Q
and B. Lastly, absolute pass/fail values for the test were established that
correspond to each detector´s resolution capabilities.
278/P132
A FLEXIBLE AND EXTENSIBLE ARCHITECTURE FOR
VISUAL WORKFLOW DEVELOPMENT
Jaeick Oh1
1
Image-based high-content analysis involves a complex series of tasks
including image acquisition, image analysis, and data analysis. The
development of diverse and sophisticated assays often requires
customizing many steps of data acquisition and analysis. The iGeneration
Cytometric Analysis Software has a flexible and extensible architecture
that allows users to customize and augment any stage of assay
development, using a visual programming paradigm. Visual
programming allows users to create workflows by connecting modules,
166
Mel Henriksen1
1
Quantitative Imaging Cytometry (QIC) is a hybrid discipline merging
the quantitative capabilities of laser-based systems with the spatial
resolution capabilities of camera-based systems. While there are no
hard lines drawn between instrument platforms regarding types of assays
that can be performed, each technology has its own nuances and areas
of advantage. The proposed platforms to be evaluated will include
Flow Cytometry, Laser Scanning Cytometry and Camera-Based imaging
systems. These platforms utilize a range of excitation and collection
technologies such as lasers and arc lamps, and cameras and PMT systems,
using both confocal and collimated light. We will describe key areas of
comparison in deciding the appropriateness of different technologies,
including: • Analytical performance such as sensitivity, precision, and
spatial resolution • The ability to perform multiplexed assays on a single
platform • The ability to analyze a variety of sample types on a single
platform. We propose three model systems to be used for these
evaluations: • Single- and multi-level calibration particles to assess
analytical performance • Adherent cells to assess real-world assay
performance and workflow for biomedical R&D • Tissue sections /
TMAs to assess performance and workflow in this newly emerging and
critical area of biomarker discovery and cancer therapeutics.
280/P134
INSTRUMENT CALIBRATION FOR DETERMINATION
OF RELATIVE FLUORESCENCE INTENSITY ON
POLYCHROMATIC FLOW CYTOMETERS
Constance Porretta1, Judd Shellito1, Steve Nelson1, Ping
Zhang1
1
LSU Health Sciences Center, Dept. of Medicine, Section of
Pulmonary/CCM, New Orleans, Louisiana
Polychromatic flow cytometers such as the FACSAria present unique
challenges when employed for comparing relative fluorescence intensity
of multiple parameters in longitudinal studies. Fluorescence area (FLA) is a calculated value that can be affected by variations of instrument
conditions including sample pressure, flow rate, and nozzle position.
These instruments are optimized on a daily basis for laser delay and area
scaling. Further optimization is recommended for each sample or
experiment using biological controls, e.g. unstained or isotype-labeled
cells. Daily instrument settings can vary significantly, which alters the
fluorescence intensity determined from a given marker. This situation
complicates day-to-day analysis of up- or down-regulation of receptors
or antigens. We have observed wide discrepancies in determined
fluorescence intensity of fluorochrome-labeled antibodies bound to
CompBeads on different days after instrument “optimization.” In order
to minimize the effect of daily instrument setting variations, we have
performed a series tests using a FACSAria flow cytometer and developed
a protocol. Laser delay and area scaling are set using Sphero Rainbow
particles as recommended by the manufacturer. Sample optimization is
performed and PMT voltages adjusted to give desired signal/noise ratios
for each fluorochrome. Optimal sample flow rate is selected. Rainbow
particles are acquired again with the sample-optimized instrument
settings. Histograms of FL-A for each PMT are plotted and a tight
region drawn on each peak. This template is then used for calibration of
signal intensity on subsequent daily measurements. Our data indicate
that this procedure is practical for instrument calibration when comparing
relative fluorescence intensity of multiple parameters is required for
longitudinal studies.
283/P137
VARIABILITY OF OPTICAL FILTER SIGNAL TO NOISE
RATIOS IN THE RED EMISSION SPECTRUM FOR FLOW
CYTOMETRY
Lindsey Laycock1, Gayle Thornbury1, Gary De Jong2
1
281/P135
CONCEPT FOR THE TRACEABILITY OF
FLUORESCENCE (BEADS) IN FLOW CYTOMETRY:
EXPLOITING SATURATION AND MICROSCOPIC
SINGLE MOLECULE BLEACHING
Jörg Neukammer1, Carsten Gohlke2, Benedikt Kraemer3,
Martin Roos4
1
Physikalisch-Technische Bundesanstalt, Berlin, Germany; 2LINOS
Photonics SARL, Champangne au Mont d’Or, France; 3PicoQuant,
Berlin, Germany; 4Robert Koch-Institut, Berlin, Germany
We have determined the fluorescence yield of stained micro beads, used
for calibration purposes in flow cytometry, as function of the irradiance
of the exciting laser beam. A rate equation model has been applied to
derive the number of fluorescence molecules carried by each micro
bead. To derive in situ photo-physical properties of the specific dye,
required for the rate equation model, we discuss an approach based on
flow cytometric sorting of micro beads, which have passed two laser
beams with properly chosen different irradiances, and subsequent
observation of single molecule bleaching employing high sensitivity
microscopy. The feasibility of our approach is demonstrated presenting
first results concerning saturation of fluorescence of beads in flow and
single molecule bleaching by high sensitivity microscopy.
282/P136
CRITICAL ANALYSIS OF FLOW CYTOMETER
LINEARITY AND METHODS USED TO ASSESS IT
Robert A. Hoffman1
1
Linearity for quantitative measurements is defined as strict
proportionality. Deviation from linearity may have obvious effects,
such as the G2/M population in a DNA histogram being more or less
than 2.00 times the G0/G1 population. Or the effect may be subtle, such
as errors in fluorescence spectral overlap compensation where the
mathematics presumes the data are linear. For most purposes, a high
degree of linearity is also required over the large dynamic range of flow
cytometry measurements (typically 4 decades). For many users of flow
cytometry, the primary concern with “linearity” has been in regard to
the accuracy of logarithmic amplifiers. More recent generations of
instruments use wide dynamic range analog-to-digital converters, which
are inherently linear over most of their operating range. The easiest way
to test for linearity is to run a sample containing a mixture of particles of
known relative intensities. But this approach is only as reliable as the
relative intensity values assigned to the particles. A dual pulse method
using either light pulses from a light emitting diode (ref.1) or pulses
from a mixture of two different intensity fluorescent beads (ref. 2) can
be used to measure deviation from linearity. The dual pulse method
was used to confirm that a reference flow cytometer´s fluorescence
measurement deviated from linearity by less than 1% over a 3 decade
dynamic range. Various sets of beads were analyzed on the reference
instrument to determine their relative intensities, and the results were
compared to the manufacturer´s values. NIST FITC beads RM 8640
were linear within the uncertainty range provided by NIST. But according
to my results, better estimates of the two dimmest beads would be 1250
and 5015 MESF compared to the NIST reference values of 1100 and
4700 MESF. No other provider of bead sets gives uncertainty limits for
the assigned values. A sample of BD QuantiBRITE™ beads had
maximum deviation from linearity of 3% using a best fit of
proportionality. Details of the dual pulse calibration for the reference
instrument will be provided along with data for other bead sets sold to
assess linearity. REFERENCES 1. Hamatasu Photonics K.K. (1994).
Photomultiplier Tube: Principle to Application. Handbook published
by Hamatasu Photonics K.K. pp. 40-41. 2. Bagwell, C. B., Baker, D.,
Whetstone, S., Munson, M., Hitchcox, S., Ault, K. A. and Lovett, E. J.
(1989). A simple and rapid method for determining the linearity of a
flow cytometer amplification system. Cytometry 10, 689-94.
BC Cancer Agency, Terry Fox Laboratory, Vancouver, British
Columbia, Canada; 2British Columbia Cancer Agency, Vancouver,
British Columbia, Canada
Optical filters used in flow cytometry are not all created equal. Quality
varies from lot to lot and between manufacturers, and over time, filters
degrade due to exposure to high energy laser light, improper handling
and cleaning. Eleven emission bandpass filters ranging in the detection
of 650nm and 690nm (red) light were compared on BD’s FACSVantage
SE and Cytopeia’s Influx analyzer. The emission of both CD45 APC
stained cells and APC labeled CaliBRITE beads were compared in terms
of Signal to Noise ratio (S/N). The Influx analyzer reported on average
slightly lower S/N values as compared to the Vantage SE which may be
due to the power output of the lasers (25mW/35mW). S/N values ranged
from approximately 5 to 100 with elevated S/N values with beads as
compared to cells. The results indicate that S/N ratios seen with beads do
not necessarily correlate with the results seen when using cells.
Furthermore, a high positive signal does not reflect a high S/N ratio
which indicates that a low background signal is an important feature in
quality optics. S/N ratios significantly vary which affects the apparent
Stokes shift and consequently the ability to separate negative and positive
populations. The importance of regular testing of optical filter
performance is essential to ensure quality and consistent cell analysis
and cell sorting.
284/P138
EFFECTS OF INTRINSIC INTER-INSTRUMENT
VARIATION ON QUANTITATIVE FLOW CYTOMETRIC
CALCULATIONS
Ryan Duggan1, David Leclerc1
1
University of Chicago, Flow Cytometry Facility, Chicago, Illinois
Within inter-institutional collaborations and in institutions with multiple
flow cytometry platforms, standardization of quantitative experiments
across platforms has become important. The concept of Molecules of
Equivalent Soluble Fluorochromes (MESF) has been used in flow
cytometry to provide tools to compare data quantitatively over time
and across platforms. A lot of effort has been made to monitor the effect
variations of environmental changes on the fluorochromes and biological
standardization of samples : however, the characterization of the
variability of inter-instrument analysis due to optical properties or
photoelectric signal processing has yet to be elucidated. We hypothesized
that a significant part of the variance associated with the calculation of
MESF value of a given sample between different instruments is due to
the intrinsic properties of these instruments. To determine these interinstrument differences, we used Quantum FITC MESF beads (Bangs
Laboratories Inc.) to obtain the MESF value of FITC Spectracomp
beads (Dako) on five benchtop analysers within our core facility : Dako
Cyan ADP, BD FACScan, two different BD FACSCantos and a BD LSRII.
We observed a significant difference in the MESF values obtained on
the Cyan ADP and the FACScan when compared to the two FACSCantos
and the LSRII. Future experiments will allow us to determine the influence
of quantum efficiency and backgroud noise of each instrument on the
MESF value as well as the effect of varying the detector voltage to
determine whether the differences observed can be “corrected” optically
(impacting on the sensitivity of the detection) or mathematically using
software correction.
285/P139
DEVELOPMENT OF AN INTERSOCIETY LABORATORY
FLOW & IMAGING DATA EXCHANGE SPECIFICATION
AND/OR STANDARD
Robert CARY Leif1
1
Newport Instruments, Research & Development, San Diego,
California
Objective: Creation of a widely supported standard for both flow
cytometry and digital microscopy. In order to achieve wide support,
the development of a common standard for scientific and clinical use
requires multiple societies and other organizations to pool their efforts.
167
Two of these societies are ISAC, which has FCS (Flow Cytometry
Standard), and the Association for Pathology Informatics, which has
started on the Laboratory Digital Imaging Project (LDIP) Data Exchange
Specification (http://www.ldip.org/). One group, the Open Microscopy
Environment (OME) (http://www.openmicroscopy.org) has already
created XML schema and software that could be used in a standard. The
Digital Imaging and Communications in Medicine (DICOM) Working
Group 26 is proposing to develop an extention for cyto and
histopathology. The Flowcyt project (http://www.flowcyt.org/) has started
work on development of a gating standard for flow cytometry and
sorting, and CytometryML. Methods: Use the domain knowledge of
the participating societies and groups, which includes detailed knowledge
of existing standards, to create a common vocabulary of elements,
attributes, and data-types, as well as definitions, which will then be
incorporated into or referenced by XML schemas. Results: The feasibility
of creating these schemas has been demonstrated by the previous
independent creation of the CytometryML schemas and the OME
software system. The CytometryML schemas describe instruments,
staining, and data. Many of the data types are based on the Digital
Imaging and Communications in Medicine (DICOM). Binary files for
images and list-mode data have been created and read. OME includes
schemas and a set of tools for an image database and interface system to
store image data from multi-dimensional images from biological
microscopes and imaging systems. Conclusions: A common data
specification for digital microscopy and flow cytometry can be created
in a manner consistent with its use for medical devices and interoperability
with both hospital information and picture archiving systems. This
specification and/or standard will allow research and pathology images,
list mode data, and accompanying annotations to be exchanged, stored
in databases, and facilitate the creation of software by third parties.
286/P140
PROPOSED GATING STANDARD FOR FLOW
CYTOMETRY
Josef Spidlen1, Robert Gentleman2, Perry Haaland3, Michael
F. Ochs4, Charles Schmitt5, Clayton Smith6, Adam S Treister7,
Ryan R. Brinkman8
1
British Columbia Cancer Research Centre, Terry Fox Laboratory,
Vancouver, British Columbia, Canada; 2Fred Hutchinson Cancer
Reserch Center, Seattle, Washington; 3BD ViaSante, Carrboro,
North Carolina; 4Fox Chase Cancer Center, Philadelphia,
Pennsylvania; 5BD Technologies, Research Triangle Park, North
Carolina; 6Terry Fox Laboratory, BC Cancer Research Centre,
Vancouver, British Columbia, Canada; 7Tree Star, Inc., Redwood
City, California; 8British Columbia Cancer Research Centre,
Vancouver, British Columbia, Canada
Gating in flow cytometry is a well known and highly important process
for selecting populations of interests by defining the characteristics of
particles for further data acquisition or analysis. It may also be used for
sorting purposes, e.g., for distinguishing among multiple heterogonous
populations in a single sample.Although flow cytometry has a successful
data format standard (FCS files), there is no shared representation of
gates. This prevents a variety of collaborative opportunities to recreate
experimental methods and results.Several partners from academy as
well as from industry joined within the project NIH R01 EB-5034 to
collaborate on the development of data standards in flow cytometry. We
have developed a proposal on how to form XML-based gate definitions
that can facilitate the interchange and validation of data between different
software packages. It currently consists of four parts as follows:
1. A detailed description of the gating specification.
2. A W3C schema document defining the syntax of XML
documents describing gates. The schema can also be used to
validate them.
3. A comprehensive user documentation presenting the schema
in a lucid manner.
4. A set of examples of gating XML files.
The specification currently supports rectangular gates in n dimensions
(i.e., from 1D range gates up to n -dimensional hyper-rectangular
regions), polygon gates in 2 (and more) dimensions, ellipsoid gates,
decision tree structures, and Boolean collections of the any of the types
168
of gates. We also provide mechanisms to link the gating file to a particular
FCS file, to express transformations of dimensions, or to express fuzzy
gates.Moreover, we are preparing a platform independent open source
software tool that is capable of reading gate definition files, applying
them to specified FCS data files, and providing common descriptive
statistics for selected parameters. We plan to release this tool along with
the gating specification in order to provide a freely available reference
implementation for independent software developers.The draft of the
gating standard has progressed through several iterations among the
project collaborators and is now being presented to the broader ISAC
community with a kindly request for comments. The most up-to-date
version can be downloaded from the project web site at www.flowcyt.org.
287/P141
EVALUATION OF THE EFFECTS OF ERYTHROPOIETIN
ON ERYTHROID PRECURSOR POPULATIONS IN
MURINE BONE MARROW USING A PATTERN
RECOGNITION APPROACH
Ram Achuthanandam1, Renold J. Capocasale1, John Quinn2,
Peter Bugelski3, Leonid Hrebien4, Moshe Kam5
1
Centocor Inc., Radnor, Pennsylvania; 2Drexel University,
Biomedical Engineering, School of Biomedical Engineering,
Science, and Health Systems, Philadelphia, Pennsylvania;
3
Centocor, Toxicology and Investigational Pharmacology/
Experimental Pathology, Malvern, Pennsylvania; 4Electrical And
Computer Engineering Department, Drexel University, Philadelphia,
Pennsylvania; 5Drexel University, Electrical & Computer
Engineering, College of Engineering, Philadelphia, Pennsylvania
Recombinant human erythropoietin (rh-EPO) has been found to stimulate
expansion of BFU-e and CFU-e in cell culture. Only recently, the in
vivo effects of rh-EPO on proliferation and maturation of late stage
erythroid precursors in mice have been studied. Our study focuses on
the effects of rh-EPO on late stage erythroid precursor populations
using a non-subjective statistical approach. Four-color flow cytometry
was used to analyze a rh-EPO dose response on erythroid (Ter-119 +,
CD71) and non-erythroid populations (myeloid: Gr-1 +, CD-11b +, Blymphocyte: IL-7Ra + , stem cell: Sca-1) from murine bone marrow.
Subcutaneous rh-EPO was administered at 6 dosage levels (30, 100,
300, 1000, 3000 and 10000 U/kg) to C57Bl/6 mice (4 mice/group)
while corresponding age matched controls received phosphate buffered
saline (pbs). A statistical data analysis approach was used to counter
some of the bias inherent with current flow cytometric data analysis
methods. A modified version of the Kolmogorov-Smirnov (KS) twosample test was used. Additionally, the Robust Competitive Agglomerative
Clustering (RCA) algorithm was used to determine the presence of
distinct bone marrow subpopulations that responded to rh-EPO. We
were able to identify regions in six-dimensional flow cytometric data
consistent with late stage erythroid precursors. Rh-EPO was not found
to stimulate any of the non-erythroid lineage populations in the bone
marrow. RCA clustering resulted in identification of four subpopulations
within the late stage erythroid precursors. Cell sorting resulted in
identification of subpopulations as follows: early proerythroblasts
(ProEB), early basophilic erythroblasts (EBasoEB), late basophilic
erythroblasts (LBasoEB), polychromatophillic and orthochromic
erythroblasts (POEB). In conclusion, rh-EPO drives late stage erythroid
precursor population expansion, dose dependently in murine bone
marrow. The four subpopulations identified respond differently to rhEPO. Greatest levels of expansion were observed in the EbasoEB
subpopulation. Taken together these methods reveal differential effects
in morphologically distinct erythroid subpopulations with rh-EPO dosage.
Identification of biologically significant regions, including identification
of subpopulations, can thus be performed using a statistical method
without prior biological knowledge in an objective manner.
288/P142
FULL AUTOMATION OF FLUORESCENCE
COMPENSATION AND EXTRACTION OF ADDITIONAL
INFORMATION OF VALUE FOR IMPROVING MULTICOLOR FACS ANALYSIS
290/P144
COMPARISON OF MODEL PROTEIN QUANTIFICATION
USING FORWARD-PHASE PROTEIN MICROARRAYS
AND SUSPENSION ARRAYS
David Parks1, Wayne A. Moore2
Lili Wang1, Kenneth Cole1, Hua-Jun He1, Diane Hancock1,
Yaping Zong2
1
1
Herzenberg Lab, Stanford, California; 2Stanford University,
Genetics, Stanford, California
The steps in preparing multi-color flow cytometry data for biological
analysis, particularly specification of fluorescence compensation, can
be time consuming and prone to error or inconsistency in control sample
gating. We report progress in the development of a new method for
evaluating fluorescence compensation based on fitting a comprehensive
data model for samples stained with a single dye. This has the advantage
over current methods of requiring no user intervention and providing
quality assurance information including error estimates for the fit as a
whole and for the spectral overlap/compensation matrix elements
themselves. This information will give confidence that the results of the
automated analysis are reliable (or alert users to problems with the data
provided) and will diagnose several kinds of FACS instrument problems
that impact data quality, such as non-linearity in electronics and laser
misalignment in multi-laser systems. We have developed robust methods
for preliminary fitting, data pruning and final fitting of the model to
particular data sets. Besides specifying the best estimate spectral overlap
matrix we obtain the photoelectron scale for each data dimension.
Knowing photoelectron scales makes it possible to perform a valid chisquare test on the fit and to estimate “fluorescence-minus-one” (FMO)
control distributions without preparing and analyzing actual FMO control
stains. Standard FMO controls are important tools for evaluating cell
populations that are not well resolved from background, but they have
to be planned beforehand and use up cells, reagents and other resources.
289/P143
APPLICATION OF TEMPORAL TEXTURE FEATURES TO
AUTOMATED ANALYSIS OF PROTEIN SUBCELLULAR
LOCATIONS IN TIME SERIES FLUORESCENCE
MICROSCOPE IMAGES
Yanhua Hu1, Jesus Carmona2, Theodore SCOTT Nowicki1,
Robert F. Murphy3
1
National Institute of Standards and Technology, Biochemical
Science, Gaithersburg, Maryland; 2Full Moon BioSystems, Inc.,
Sunnyvale, California
Protein microarrays, one emerging class of proteomic technologies,
have broad and yet unique applications for quantitative analysis and
discovery. Compared to other proteomic technologies, such as 2-D gel
electrophoresis combined with mass spectrometry, suspension arrays,
micro-ELISA, and multiplexed immunoassays, protein microarrays have
unique capabilities for network profiling of cellular clinical samples. At
the present time, quantitative protein analyses with the use of protein
microarrays are carried out by means of a serial dilution of the analytes.
There are a limited number of comparative studies focusing on the
quantification aspects of the proteomic methods based on the use of
antibody-antigen interactions. Knowledge of the advantages and
disadvantages of these different formats would allow their more rational
use in clinical diagnosis. In this study, we employed ovalbumin (a
simulant used for ricin and botulism toxins in biodefense applications)
and its high affinity polyclonal antibody as a model system. We used
this system to examine the reliability, sensitivity, dynamic range, and
linearity of forward-phase array results with respect to suspension arrays.
It was found that protein arrays had a linearity of no less than 3 orders
of magnitude and a sensitivity of about 0.5 pg/mL, respectively. The
linearity and sensitivity of suspension arrays were close to 2 orders of
magnitude and 0.25 ng/mL, respectively. The sensitivity we observed
for the suspension arrays is comparable to that reported for ELISA-type
assays reported in the literature. We used ovalbumin samples with two
different purities, 38.0% and 75.8% (w/w), as determined by
polyacrylamide gel electrophoresis (PAGE). These samples were used
to test the effect of impure samples on detection. The data obtained
from the forward-phase protein arrays gave values that were consistent
with the PAGE data. The data from the suspension arrays was not as
consistent and may indicate this format may not give as reliable data
with impure samples.
Carnegie Mellon University, Center for Bioimage Informatics,
Department of Biological Sciences, Pittsburgh, Pennsylvania;
2
Carnegie Mellon University, Center for Bioimage Informatics,
Department of Biomedical Engineering, Pittsburgh, Pennsylvania;
3
291/P145
INTEGRATION OF FLOW CYTOMETRY TECHNOLOGY
INTO A MASS SPECTROMETRY BASED PROTEOMICS
PLATFORM
The subcellular locations of proteins are commonly determined by
fluorescence microscopy. Previous work by our group has shown that
automated analysis of static 2D and 3D images can recognize all major
subcellular patterns, and that automated methods can be used to
distinguish patterns that are subtly different. Since many proteins are in
constant movement within the cell, we extended our studies to time
series images, which contain both spatial and temporal information. In
the general case, protein movement is hard to analyze using tracking
methods for two reasons: first, proteins in many cases are not well
grouped into objects that can be used as a target for tracking; second,
there is often no ground truth to allow accurate evaluation of automated
tracking methods. To bypass these problems, we have used a set of
temporal texture features, which do not require predefining protein
objects, to represent changes in protein distribution over time. The
temporal texture features we used are modified from the classic Haralick
texture features on static images, and describe the intensity relationship
of neighboring pixels in time. We used two methods to evaluate the
movement information captured by temporal features. We first compared
the ability of automated classifiers to distinguish a set of five 3T3 cell
lines expressing GFP-tagged proteins with similar patterns, and found
that overall accuracy of discrimination was increased from 75% using
features calculated on static images to 85% with temporal texture features
added. We also made automated comparison of protein locations in
pairs of tagged cell lines expressing and not expressing the h-Ras
oncogene that were created in Dr. Jonathan Jarvik´s laboratory. By
adding the temporal texture features, we were able to distinguish one
pair of these lines that was previous considered to be indistinguishable
in static images. We conclude that temporal textures are a simple yet
powerful tool for distinguishing protein subcellular location patterns.
1
Katherine McKinnon1, Christine Evangelista1, Elizabeth
Joseloff1, Sudeepta Aggarwal1, Tao He1, Anne Deslattes
Mays1, Paul Moore1, Charles Birse1, Steve Ruben1
Celera Genomics, Rockville, Maryland
The successful application of therapeutic monoclonal antibodies in the
treatment of a variety of cancers has stimulated the search for additional
tumor antigens. Using a Mass Spectrometry (MS) based proteomics
platform we have identified a panel of cell-surface and secreted proteins
upregulated in several solid tumor indications. Flow cytometry is utilized
at several stages in our proteomic process and makes major contributions
to this analysis at both the target identification and validation stages.
The proteomic process begins with the enzymatic/mechanical breakdown
of fresh tissue samples into a single cell suspension followed by flow
cytometric characterization of each sample. This assessment provides
an analysis of the cell composition (epithelial, endothelial, immune,
stromal) and cellular viability, ultimately determining the fate of the
specimen. Cell populations selected for proteomic analysis by MS are
purified by cell sorting and enriched for cell surface proteins. As such,
flow cytometry provides a means of selecting multiple cell populations
of interest for target discovery. Once the differentially expressed proteins
have been identified by MS, they undergo a validation process that
consists of functional analysis (RNAi, functional antibodies) and
expression analysis (IHC, TaqMan and flow cytometry). Initial flow
cytometric validation consists of expression analysis of the targets in
both tissue and cell lines; this serves to both extend the expression
profile and identify appropriate cell lines for subsequent functional
studies. Subsequent quantitative flow cytometry analysis is performed
on tissues, blood and bone marrow to further evaluate the specificity
and copy number of this target. Where appropriate, flow cytometry is
used to support RNAi validation studies, and in a series of proliferation,
169
apoptosis and phosphorylation assays to evaluate the possible role of
targets in tumor progression. Data will be presented exhibiting how the
analysis and sorting capabilities of flow cytometry play a central role in
our proteomics laboratory.
292/P146
EFFICIENT DELIVERY OF SIRNA INTO DIVERSE CELL
TYPES WITH LOW TOXICITY VIA LASER-MEDIATED
OPTOINJECTION
Kate Rhodes1, Imran Clark1, Michelle Zatcoff1, Trisha
Eustaquio1, Kwame Hoyte1, Manfred Koller1
1
Cyntellect, San Diego, California
Since the recent discovery of effective small interfering RNA (siRNA)mediated gene silencing via RNA interference (RNAi) in mammalian
cells, there has been significant validation and enormous interest in the
approach from both academic and corporate researchers, for a variety
of discovery and therapeutic applications. However, delivery of siRNA
into many important cell types remains problematic due to poor efficiency,
cell toxicity, and/or physiological changes that are induced by current
transfection methods such as cationic lipids and electroporation. To
address these issues, a novel laser-based cell transfection approach was
developed. An automated high-throughput instrument, the LaserEnabled Analysis and Processing (LEAP™) system, was utilized to
elucidate and optimize several parameters that influence optoinjection
efficiency and toxicity. Techniques employing direct cell irradiation
(i.e., laser targeted to specific cell coordinates calculated based on image
analysis) and grid-based irradiation (i.e., laser targeted to fixed grid
coordinates without locating cells) were both successfully developed.
With both techniques, it was determined that multiple, sequential low
radiant exposures produced more favorable results than a single high
radiant exposure. The success of the grid-based irradiation approach
led to development of a new instrument (called HOP™ for Highthroughput OPtoinjector) that is significantly simpler and smaller than
LEAP. High-efficiency, laser-mediated delivery of siRNA was
demonstrated in a number of recalcitrant cell types (e.g., human B- and
T-cells, neurons) on both LEAP and HOP, in each case with less cell
toxicity than standard methods. Laser-mediated transfection of siRNA
was shown to be consistent across many cell types, enabling RNAi
studies to be conducted in difficult-to-transfect cells. Finally, evidence
is provided to suggest that the mechanism of optoinjection employed
here is distinct from the hole-punching and shock wave mechanisms
that have been proposed in prior reports of optoinjection.
293/P147
ULTRASENSITIVE SINGLE-CELL AND POPULATIONLEVEL ANALYSES OF PROTEIN EXPRESSION IN
DEINOCOCCUS RADIODURANS BY CAPILLARY
ELECTROPHORESIS WITH LASER-INDUCED
FLUORESCENCE
Emily Turner1, Norm Dovichi1
1
University of Washington, Chemistry, College of Arts and
Sciences, Seattle, Washington
Heterogeneous responses of isogenic bacterial populations to external
stimuli are well documented. Traditional biochemical analyses mask
stochastic protein expression by averaging across the population. For
comprehensive determination of the relationship between protein
expression and cellular response, individual cells must be analyzed.
Our group has developed capillary electrophoresis with laser-induced
fluorescence for rapid detection of fluorescently-tagged cellular proteins.
This ultrasensitive technology routinely detects proteins at the subzeptomole level (1 zmol = 10 -21 mol), and is capable of detecting a
single green fluorescent protein (GFP) molecule. Using the same capillary
electrophoresis instrument, protein expression in Deinococcus
radiodurans is being determined at the single-cell and population
levels. D. radiodurans is the most radioresistant organism yet
discovered, and shows extraordinary capacity to repair DNA. The RecA
protein has been identified as key to the DNA repair mechanism in D.
radiodurans. Using a RecA-GFP fusion construct, analysis of a potential
adaptive response to DNA damage is being performed with capillary
electrophoresis. To analyze single cells, a bacterium of interest is
identified by fluorescence microscopy. The cell is injected into the
capillary, where chemical lysis occurs. Cellular proteins are separated
170
across the capillary by the application of high voltage, resulting in rapid
detection of released GFP. For population analysis by capillary
electrophoresis, intact cells are continuously injected into the capillary,
and whole-cell fluorescence is detected. Development of this technology
will enable rapid and ultrasensitive analysis of any fluorescently-tagged
protein in single prokaryotic or eukaryotic cells.
294/P148
DATA QUALITY ASSESSMENT IN FLOW CYTOMETRY
EXPERIMENT
Nolwenn Le Meur1, Robert Gentleman1, Maura Gasparetto2,
Clayton Smith2, Ryan R. Brinkman2
1
Fred Hutchinson Cancer Research Center, Computational Biology,
Seattle, Washington; 2British Columbia Cancer Research Center,
Terry Fox Laboratory, Vancouver, British Columbia, Canada
The recent development of semi-automated techniques for staining and
analyzing flow cytometry samples has presented new challenges.
Standardization is critical when developing new high throughput
technologies and their associated information services. Our experience
suggests that data quality control and data quality assessment, not yet
systematically applied, are crucial steps in the development standards in
high throughput flow cytometry data. The aim of data quality assessment
is to detect to detect systematic and stochastic effects that are not likely
to be biologically motivated. The rationale is that systematic errors
often indicate the need for adjustments in sampling handling or
processing. Further, the aberrant samples should be identified and
potentially removed from any downstream analyses in order to avoid
spurious results. Data quality assessment in high throughput flow
cytometry experiment is complicated by the volume of data involved
and by the many processing steps required to produce those data. We
propose a variety of exploratory data analytic (EDA) tools, mainly
graphical methods, for exploring the data in a time and cost effective
manner. We rely mainly on the R programming language for our basic
statistical tools and have implemented a number of specialized functions
and methods in the BioConductor package rflowcyt. We demonstrate
the use of these approaches by investigating two independent sets of
high throughput flow cytometry data and showing substantial nonbiologically motivated differences in samples, that went undetected
during manual review of the high throughput data.
295/P149
PROTEOMIC STRATEGIES TO ANALYZE CELL-FREE
FRACTIONS FROM ACTIVATED PERIPHERAL BLOOD
MONONUCLEAR CULTURES
Sybil S. D’Costa1, Julie G. Wilkinson1, Enrique Rabellino1
1
Beckman Coulter Inc., Custom Biopharma Solutions, Miami,
Florida
The use of Proteomic strategies in the discovery process is imperative
since post-transcriptional modification can produce dramatic changes
in protein levels and activity that are invisible to DNA arrays. The
introduction of new and improved proteomics solutions with increased
sensitivity, specificity and ease of use has been integral in facilitating
this process. The current study has evaluated signatures of immune
response in cell-free fractions of control and activated peripheral blood
nuclear cell cultures using proteomic combinations designed to improve
sensitivity of detection and ease of use. Peripheral blood mononuclear
cells cultured in medium containing human AB serum were subjected to
activation for 24hrs using Staphylococcal enterotoxin B. Cell-free
fractions from the activated and control cells were fractionated by twodimensional chromatography in the liquid and intact phase. To improve
the sensitivity of detection of protein signatures, the secreted components
were also subjected to a fractionation strategy using IgY antibodies to
deplete the most abundant proteins in human serum and then analyzed
by two-dimensional liquid chromatography. Intact proteins were
separated by their isoelectric points in the first dimension and further
separated by hydrophobicity on a second-dimension. The net result was
the generation of high-resolution protein profile of the complex mixture.
Qualitative and quantitative differences in protein profiles in activated
and non-activated cell free fractions could be easily identified using
powerful software. The use of the IgY fractionation technique to deplete
the abundant proteins in serum containing growth medium dramatically
enhanced the sensitivity of the differential analysis. The gel- free and
intact nature of the fractions of interest allows for further interrogation
and identification of the differentially expressed proteins to elucidate
an activation signature in supernatants. Thus the combination of
proteomic techniques enables a more refined and targeted profiling and
analysis of complex events associated with an immune response
296/P150
IDENTIFICATION OF IMMUNE RESPONSE
SIGNATURES UTILIZING INTEGRATED CYTOMIC AND
PROTEOMIC TECHNIQUES
Sybil S. D’Costa1, Julie G. Wilkinson1, Enrique Rabellino1
1
Beckman Coulter, Inc., Custom Biopharma Solutions, Miami,
Florida
The identification of signatures associated with disease could impact
every aspect of patient care from screening to treatment. To arrive at
such signatures a multipronged approach needs to be pursued. Studies
that have so far been carried out in isolation for e.g. gene expression,
protein synthesis, etc. need to be integrated to understand the complex
responses that translate to patterns. In the current study the authors have
attempted such an evaluation by integrating well known techniques of
cell sorting with protein fractionation to identify signatures of immune
cellular activation. Peripheral blood mononuclear cells were subjected
to restricted polyclonal stimulation with staphylococcal enterotoxin B
(SEB) or left untreated. Cells were then labeled to identify T cells and
isolated using flow-based sorting techniques. Lysates of unsorted
activated and non-activated PBMCs as well as sorted T cells were
fractionated by two-dimensional gel-free liquid chromatography. Intact
proteins were separated by their isoelectric points in the first-dimension
and further separated by hydrophobicity on a second-dimension. Using
powerful differential display software a high resolution protein profile
of the complex mixtures was obtained. Qualitative and quantitative
differences in protein profiles in activated and non-activated cells were
identified using a “proteomic only” strategy resulting in immune response
signatures. Further integration of cell sorting techniques with proteomics
refined the profile by enabling a finer mapping of the T cell signatures.
The liquid-phase fractions of proteins in the intact state, permits direct
characterization of the differentially-expressed proteins. Further
combinations of genomic, proteomic and cytomic profiling will
accomplish a unified evaluation of signatures relevant to this response
and other research models.
297/P151
CANCER RELATED CANDIDATE GENES SELECTION
AND MANUFACTURE THE FUNCTIONAL OLIGO
MICROARRAY OF ORAL SQUAMOUS CELL
CARCINORMA
Liqiong Duan1, Wan-Tao Chen1, Ping Zhang2, Xiaozhi Lu3,
Ming Yan3, Yan Lu3, Jie He3, Zhiyuan Zhang3
1
Shanghai Second Medical University Affiliated Ninth
Hospital,Shanghai Key Lab of Stomatology, Shanghai, 200011,
China; 2Shanghai Second Medical University Affiliated Ninth
People’s Hospital,Shanghai Key Lab of Stomatolog, Department of
Oral and Maxillofacial Surgery, Shanghai, China; 3Shanghai Second
Medical University Affiliated Ninth People’s Hospital,Shanghai Key
Lab of Stomatolog, Department of Oral and Maxillofacial Surgery,
Shanghai, 200011, China
[Objective] : The purpose of the experiment study is to select and
identify the target genes related to oral squamous cell carcinoma (OSCC),
manufacture the oligonucleotide microarray suitable for early diagnosis,
therapeutic intervention and prognosis judgement for OSCC and find a
feasible method for individually therapeutic plans. [Method]: Cancer
related candidate Genes were selected from differentially expressed
microarray in head and neck. 5 years´ references and our previous data
were taken into account. Then these genes selected from the reference
were evaluated by Real-time Quantitative Polymerse Chain Reaction
(RQ-PCR). The mRNA expression Of selected genes were quantified in
22 cases of OSCC, including both tumor tissues and normal mucosas.
All the genes were designed according to the probe design rules of
Operon and Affymetrix database. and the 10*10mm2 functional oligo
microarray of OSCC were manufactured with all 24 selected candidate
genes. [results] : 24 genes were chosen to be our target genes ,12 genes
came from differentially expressed genechips of HNSCC in our
preliminary studies,12genes came from related references. And 59 mer
oligonucleotide probes of genes were finally vertified to be the optimal
length.60 samples (30pairs) were combined to microarray to validate
the quality and quantity of chips . [Conclusion] All the candidate genes
were closely related to tumorigenesis progress of Oral Squamous cell
carcinoma. And they can be used as the target genes for Oligo-nucleotide
functional microarry of OSCC. This study supported by National
Natural Science Foundation (30300388,30330580 )
298/P152
MICROARRAY-BASED GENOTYPING IN LARGE
POPULATIONS
David Galbraith1, Jeremy Edwards2, Ambika Gaikwad2,
Jaroslav Janda2, Stefan Schwab2, Bin Liu3, Hei Leung3
1
Tucson, Arizona; 2University of Arizona, Plant Sciences, Tucson,
Arizona; 3International Rice Research Institute, Los Banos, ,
Philippines
We report progress toward the development of a microarray platform
for rapid and cost-effective genetic mapping using rice as a model
system. Microarray-based genotyping is well suited for applications
such as Quantitative Trait Locus (QTL) mapping, where the need for
whole genome coverage can be efficiently addressed through the parallel
assay of over 1,000 genetic markers on a single microarray. One of our
major objectives is to investigate cost-saving alternative methods for
slide production and detection of the hybridized signal. The genotyping
microarrays are designed using 70mer oligonucleotide probes to detect
Single-Feature Polymorphisms (SFPs) (i.e. insertions/deletions) for use
as genetic markers. The SFPs have been identified in silico by aligning
the publicly available genomic sequences of the Nipponbare and 9311
cultivars representing the japonica and indica sub-species of rice. We
are further designing strategies for detection of Single-Nucleotide
Polymorphisms at similar levels of multiplexing, and similar costs. The
genotyping microarrays will be used for QTL mapping in the IR64 X
Azucena doubled haploid population and the SHZ X LTH recombinant
inbred line population. The genotyping data will be integrated into the
existing molecular maps of these segregating populations, enhancing
their potential mapping resolution. We are concurrently designing and
implementing experiments using microarrays for global expression
profiling to correlate the genotypes defined by the mapping microarrays
with significant changes in gene expression and phenotypic variation,
and with QTLs of agronomic importance such as resistance to disease
and abiotic stresses. The genotyping microarrays will also be used to
assay a panel of diverse rice cultivars to evaluate the potential of this
platform for applications in germplasm classification and diversity
studies.
299/P153
COMPARATIVE GENOMIC HYBRIDIZATION WITH IN
SITU SYNTHESIZED 60MER OLIGONUCLEOTIDE CGH
ARRAYS
Michael T. Barrett1, Amir Ben-Dor1, Anya Tsalenko1, Doron
Lipson1, Alicia Scheffer1, Paige Anderson1, Peter Tsang1, Bo
Curry1, Nick Sampas1, Zohar Yakhini1, Laurakay Bruhn1
1
Agilent Technologies, Palo Alto, California
Array-based comparative genomic hybridization (aCGH) is an important
tool for studying acquired and constitutive genomic variations associated
with normal populations, genetic diseases and tumorigenesis. The robust
application of aCGH to the study of human biology requires: 1)
preserving the greatest possible complexity of targets derived from
whole genome samples, 2) detection of variable lesions including single
copy changes throughout the genome in both homogeneous and
heterogeneous samples, 3) protocols that enable the use of small clinical
samples, 4) visualization and rigorous computational tools to analyze
and integrate complex data, and 5) high density array formats that can
be customized to genomes and regions of interest. We have designed
60mer oligonucleotide arrays for CGH with probes biased toward known
and predicted gene loci in the human genome. The mean slope of
experimental versus theoretical log-ratios for chromosome X probes in
male (46XY) versus female (46XX) hybridizations typically exceeds
0.9, with probe by probe error rates < 10%. The median ratios for X
chromosome probes in a series of cell lines with variable numbers (1-5)
of X chromosomes were all within 10% of the theoretical values (0.5
for 46XY/46XX, 1.0 for 46XX/46XX, 1.4 for 47XXX/46XX, 2.2 for
48XXXX/46XX, and 2.6 for 49XXXXX/46XX). Aberration calling in
171
samples of interest, including genetic syndrome and cancer cells, is
based on computing significance scores for all genomic intervals. For
an interval, J, the score represents the deviation of the sum of logRatio
values for probes in J, from its null expected value of zero. The deviation
is in terms of null expected standard deviations. We use an efficient
algorithm (ADM1) to identify all high scoring intervals. Our platform
accurately detected regions of predicted genetic loss or gain in genetic
syndrome samples, including deletion of 15q in Prader-Willi,
amplification in 17p12 for Charcot-Marie-Tooth, and deletion of a
portion of the DMD gene on Chromosome X in Duchenne´s Muscular
Dystrophy. In addition we detected somatic abnormalities, including
single and homozygous copy losses, focal amplifications, and aberrant
chromosome ploidies in a series of cancer cells and biopsies. Furthermore
we demonstrate that these lesions can be detected with small amounts of
starting sample including clinical biopsies, and in the presence of
significant sample heterogeneity. Finally we demonstrate the detection
and mapping resolution of oligonucleotide CGH arrays with genomewide coverage extended to 185,000 probes targeting all known genes
in the genome and even spacing in intergenic regions.
advantages: (1) Accuracy and rapidity (staining procedure: 5 -10 min),
because the samples are analyzed immediately on completion of
phagocytosis without any further cell manipulation; (2) The omission
of the centrifugation step at the end of the phagocytosis assay avoids
under and/or over estimations of the phagocytic process thereby giving
an accurate picture of the phenomenon; (3) Possibility of counting free,
attached and ingested yeasts; (4) Possibility for simultaneous viability
assessment of all cells. No additional bioactivitytests of every single cell
population are necessary. (5) Use of non-toxic and cheap reagents. The
method is reproducible and sensitive. The results were confirmed by
direct cell culture assay.
300/P154
DIFFERENTIAL GENE EXPRESSION IN HEPATOCYTE
SUBPOPULATIONS SORTED FROM ACETAMINOPHENTREATED MICE AND A COMPARISON OF 1 VS 3 DROP
SORTING
1
Collin C White1, Michael Dabrowski2, Carolina Fernandez2,
Richard Beyer2, Theo Bammler2, Terrance Kavanagh2
1
University of Washington, Environmental Health, School of Public
Health and Community Medicine, Seattle, Washington; 2University
of Washington, Environmental Health, seattle, Washington
Acetaminophen overdose is associated with acute liver injury
characterized by centrilobular necrosis. This is thought to occur because
APAP is activated to a reactive intermediate by cytochrome P450 enzymes
present in centrilobular hepatocytes. In order to determine whether the
sensitivity of detecting gene expression changes is enhanced by
examining specific cell types with an organ, we used fluorescence
activated cell sorting to isolate different hepatocyte subpopulations from
the liver of APAP treated mice. Fasted mice were treated with APAP
(300 mg/kg BW) or saline and 6 hr later, viable hepatocytes were
isolated via collagenase perfusion. Isolated hepatocytes were stained
for mitochondrial membrane potential using JC-1 and live cells were
sorted into subpopulations of high JC-1 staining and low JC-1 staining
cells. In addition these subpopulation were sorted with both 1 and 3
drop sorting. Total RNA was then extracted and subjected to DNA
microarray analysis using the Affymetrix platform. APAP treatment
affected the expression of a large number of genes in each of the two
JC-1 low and high staining cell populations. While APAP treatment
resulted in an overlap of differentially expressed genes in the two cell
populations, each population also exhibited its own specific profile of
differentially expressed genes. Cell populations collected by a 1 drop
sort resulted generally in a higher number of statistically significant
differentially expressed genes compared with the 3 drop sort, suggesting
the 1 drop sort may result in purer cell population. These data support
the hypothesis that there are unique gene expression changes in
subpopulations of hepatocytes and underscore the need to consider
such differences when interpreting gene expression profiles at the whole
organ level. Supported by NIH grants U19ES11378, P42ES04696,
303/P157
ENHANCED CYTOMETRIC BEAD ARRAY ASSAYS:
IMPROVING SENSITIVITY AND DECREASING ASSAY
TIME WITH A NOVEL AUTOMATED FLUIDICS
APPROACH
Sujata Iyer1, Julia Ember2, Brandon Bowman2, Rich M.
Ozanich3, Cindy Bruckner-Lea3, Brian Dockendorff3
Becton Dickinson, San Jose, California; 2BD Biosciences, San
Diego, California; 3Pacific Northwest National Laboratory,
Richland, Washington
An automated fluidics system in combination with a novel microparticle
trap/flow-cell was used to perform a microbead-based sandwich
immunoassay with subsequent quantitation using flow cytometry. A
five-plex cytometric bead array (CBA) assay for human chemokines
was used to evaluate the performance capabilities of the system.. Basic
assay steps included mixing five separate antibody-coupled flow
cytometry beads with chemokine samples/standards and an aliquot of
secondary fluorescent-labeled (phycoerythrin) antibody. The 3 hour
reaction period was followed by a wash step and the level of fluorescence
was measured using a flow cytometer. The fluorescence signal intensity
was proportional to the chemokine concentration. The automated system
comprised of a syringe pump, multi-position valve, control computer
and a novel microparticle trap that improved mass transport of the
target analyte, thus enhancing assay sensitivity and speed. With the aid
of larger support beads, the microparticle trap captures the smaller
antibody-coupled beads. Subsequent serial perfusion of sample/standard,
secondary antibody, and wash solution was performed, followed by
elution and collection of the beads for analysis by flow cytometry.
Potential benefits of the automated approach include (a) improved
sensitivity, (b) decreased analysis time, and (c) automation of the entire
procedure. The fluidics approach could achieve the same level of
sensitivity as the 3 hour standard benchtop procedure in approximately
one hour. Preliminary results also indicate the fluidics system can
preconcentrate target analytes from large volume dilute samples.
Approaches to optimize sensitivity and reproducibility are currently
under investigation using biological samples. The automated fluidics
platform can be readily adapted to perform other assays and can also be
directly interfaced with other sample processing and analysis equipment,
including a flow cytometer.
304/P158
EFFECT OF MICROSPHERE-BOUND SITE DENSITY ON
THE MEASURED AFFINITY OF AN INTERACTION
PARTNER
Marie Anne Iannone1, Thomas G. Consler1
302/P156
MULTI-ASSAY FOR DIFFERENTIATION OF INGESTED/
NON-INGESTED YEAST POPULATIONS AND
SIMULTANEOUS ASSESSMENT OF THE VIABILITY
1
1
Hans H. Bäuler , Verena Staedtke , Maria Fischer
1
1
Humboldt-Universität zu Berlin, Charité-Universitätsmedizin
Berlin, Berlin, Germany
Defects of the phagocytosis are responsible for several diseases of the
immune system. The evaluation of the capacity of phagocytosis of cells
includes the differentiation of cells, which adhere to the surface of the
cells and others which were ingested. We developed a flow cytometric
method to distinguish between membrane bound, ingested and free
Candida (yeast) cells. Simultaneously we can assess the viability of the
various cell populations. Our procedure provides the following
172
1
GlaxoSmithKline, Gene Expression and Protein Biochemistry,
Research Triangle Park, North Carolina
Microsphere-based binding assays using immobilized surface-bound
interaction partners can be used to detect analytes such as proteins,
nucleic acids or small molecule entities and to measure their
concentrations with high sensitivity. Multiplexed microsphere-based
binding assays may also be used to efficiently measure molecular
interactions and the effects of added small molecules on those
interactions. We have used multiplexed microsphere technology to
explore the effect that binding site density has on the apparent affinity
of a soluble interaction partner. The interaction of the nuclear receptor,
peroxisome proliferator-activated receptor gamma ligand binding
domain (PPAR gamma LBD, an important regulatory factor in lipid
metabolism and insulin sensitivity), with a synthetic peptide derived
from a nuclear receptor coactivator protein, PPAR gamma coactivator1 alpha (PGC-1 alpha), was studied. In a multiplexed experiment,
fluorescently unique microsphere populations were each coupled to the
same peptide sequence, but at different densities. The peptide sequence
is derived from the coactivator protein PPAR gamma coactivator-1
alpha (PGC-1 alpha). The peptide contains an LXXLL motif, where L is
leucine and X is any amino acid. The LXXLL signature motif tends to
form a two-turn amphipathic alpha helix which can interact with nuclear
receptor proteins along a hydrophobic groove on the receptor surface.
The density of the PCG-1 alpha peptide coupled to fluorescently unique
microsphere populations was varied by coincubating the biotinylated
peptide and avidin-coated microsphere populations with increasing the
amounts of free D-biotin. The discrete-density peptide-coupled
microsphere populations were combined to conduct a multiplexed
binding experiment with Alexa 532-labeled PPAR gamma LBD, in the
absence or presence GW1929, a small molecule ligand that has been
shown to increase the binding affinity of PPAR gamma LBD for
coactivator protein or LXXLL peptide surrogates. As the immobilized
binding site density of PGC-1 alpha peptide on fluorescent microspheres
is increased the measured apparent affinity for PPAR gamma LBD is
increased. The density of binding sites immobilized to a surface has a
marked effect on the apparent affinity for soluble binding partners. By
manipulating the binding site density it is possible to increase the
sensitivity of an interaction assay. In multiplexed assay formats it should
be possible to normalize intrinsically unequal binding interactions by
individually optimizing the binding site density of the immobilized
interaction partner. However, to quantitatively measure intrinsic affinities
of molecular interactions, low binding site densities are required and
multivalent reagents should be avoided.
305/P159
AUTOMATION OF TISSUE MICROARRAY ANALYSIS – A
NEW PARADIGM
Elena Holden1, Alexey Glazyrin2, Ed Luther1
1
CompuCyte Corporation, Cambridge, Massachusetts; 2Asterand
Corporation, Detroit, Michigan
Tissue microarrays (TMAs) have emerged as the method of choice for
evaluating clinical materials in the context of biomarker development,
for a number of reasons including the ability to evaluate large numbers
of archival tissue samples in a single experiment.
TMA preparation:
Preparation of tissue microarrays demands careful examination by a
pathologist to locate tumor areas of interest. A 360-spot TMA is typically
constructed during a five-day period by an experienced pathologist
and array technician. In this study we used a TMA containing 0.6 mm.
cores of normal breast tissue and breast adenocarcinoma. Each case was
represented by triplicate spots from tumor and normal counterpart areas.
Workflow scenarios: I. Manual pathologist evaluation: The TMA is
analyzed by an experienced pathologist on a spot-by-spot and row-byrow basis under 100x magnification, using the following steps: (1)
Overall examination with quality control for each core, to verify that
each spot represented the initial diagnosis; (2) Individual element scoring
for PR+/-, ER+/-, and HER2/Neu staining intensity (0-3); (3) Obtaining
digital camera images for representative cores and entering special
comments and results into a computer database; and (4) Data analysis
and reporting. II. Automated TMA analysis by laser scanning cytometry:
An automated analysis workflow contains the following steps: (1)
Selecting and setting the appropriate fluorescence and light absorption
signals; (2) Performing a high-speed overview scan of the TMA,
typically at 5-micron resolution; (3) Automated identification of TMA
core elements; (4) Review and editing of identified core elements; (5)
High-resolution analysis of the individual core elements; (6) Evaluation
of the results using iBrowser® Data Integration software; and (7) Data
export to a spreadsheet program. Results and conclusion: Manual
analysis of the TMA through Step 2 of the manual workflow required at
least 5-6 hours of uninterrupted, focused investigation by a trained
pathologist—a highly demanding and slow process even for experienced
practitioners. Some errors are inevitable. Automated TMA analysis is
rapid; initial scans are completed in 10–20 minutes per array. A minimal
amount of manual intervention for review and editing was required and
accomplished in 5 minutes. High-resolution scans required 1 hour of
walk-away analysis time. Automated TMA analysis by laser scanning
cytometry offers fast, objective processing with the supporting image
data archived in an accessible format. Benefits of automated scanning
warrant implementation of a new paradigm for TMA analysis.
306/P160
DESIGN AND CONSTRUCTION OF MULTIFUNCTIONAL
PARAMAGNETIC NANOPARTICLES
Mary-Margaret Seale1, Emily Haglund1, Michael D Zordan1,
Christy L Cooper2, Lisa M Reece2, Jianjie Huang3, Tarl W
Prow4, Donald Eugene Bergstrom5, James F. Leary2
1
Purdue Universtiy, Biomedical Engineering, West Lafayette,
Lafayette, Indiana; 3Purdue University, Bindley Biosciences Center,
West Lafayette, Indiana; 4Johns Hopkins University, Baltimore,
Maryland; 5Purdue University, Medicinal Chem & Molecular
Pharmacology, Pharmacy & Pharmacal Sciences, West Lafayette,
Indiana
Multifunctional nanoparticles for targeted therapeutics are being built
on 40 nm paramagnetic core nanoparticles (Miltenyi Biotec, Auburn,
CA). Dendrimeric DNA structures containing aptamers, biomolecular
sensors, tethered therapeutic genes, and fluorescent reporter genes are
being built up layer-by-layer on these nanoparticles to form multilayered
nanosystems approximately 100 nm in diameter (optimal size for in
vivo therapeutics). Since the nanoparticles are designed to disassemble
layer-by-layer in vivo, this makes them “programmable” through a
controlled sequence of events as demonstrated earlier (Leary and Prow,
US patent pending; Prow et al, 2004) on other types of multilayered
nanoparticles.
Targeting was assessed using MACS sorting,
fluorescence tracking probes, fluorescence microscopy, flow cytometry,
and LEAP™ scanning cytometry. The nanoparticle systems are designed
so that they can be heated/manipulated externally to disassemble each
layer as needed for targeting. The paramagnetic cores are designed to
provide for future multifunctional in vivo imaging and contain PCRamplifiable DNA sequences that allow them to be found even as rare
nanoparticles in tissues. This feature will be used for future biodistribution
studies in animals.
307/P161
TARGETED, MULTIFUNCTIONAL QUANTUM DOT
NANOPARTICLES FOR EX VIVO DIAGNOSTICS
Emily Haglund1, Mary-Margaret Seale1, Michael D Zordan1,
Christy L Cooper2, Lisa M Reece2, Jianjie Huang3, Tarl W
Prow4, Donald Eugene Bergstrom5, James F. Leary2
1
Lafayette, Indiana; 3Purdue University, Bindley Biosciences Center,
West Lafayette, Indiana; 4Johns Hopkins University, Baltimore,
Maryland; 5Purdue University, Medicinal Chem & Molecular
Pharmacology, Pharmacy & Pharmacal Sciences, West Lafayette,
Indiana
Multifunctional nanoparticles (NP) containing dendrimeric DNA
structures were built by layer-by-layer assembly onto core carboxylated
Qdot® (Quantum Dot Corp., Hayward, CA) nanocrystals for
development of ex vivo diagnostic systems. The motivation for this
research is to create NPs with DNA repair gene sequences to repair DNA
damage caused by radiation. This is an important medical problem
faced by astronauts traveling outside the magnetosphere for extended
periods of time.
Molecular biosensors for reactive oxygen species
(ROS), were used to detect cells with radiation damaged DNA. GFP
gene reporter sequences, downstream from the ARE biomolecular sensors,
were used to reveal presence of radiation-induced ARE (Anti-oxidant
Response Elements) molecules. Targeting accuracy was assessed by the
use of defined cell mixtures containing cell subpopulations pre-labeled
with a fluorescent membrane tracking dye. Cells were examined by
flow cytometry and LEAP™ scanning cytometry for binding and uptake
of the NPs. Three dimensional localization of NPs within single cells
was studied using confocal microscopy.
173
Figure 1. Time-dependent changes in the average sizes of
interacting RBCs and SiO2 particles of A-300.
308/P162
SIMULTANEOUS ASSESSMENT OF THREE IMMUNOPHARMACODYNAMIC PROPERTIES OF
FUNCTIONALLY DISTINCT LFA-1 ANTAGONISTS IN
WHOLE BLOOD
Karl Welzenbach1, Stephan Krähenbühl2, Gabriele WeitzSchmidt1
1
Novartis Pharma AG, Transplantation Research-ATDA, NIBR,
Basel, Switzerland; 2University Basel, Switzerland, Clinical
Pharmacology, University of Basel, Basel, Switzerland
Background: The â2 integrin LFA-1 (CD11a/CD18) is a
conformationally flexible á/â heterodimeric receptor which is expressed
on the surface of all leukocytes. LFA-1 mediated cell adhesion and
signaling events are crucial for immune responses. Allosteric low
molecular weight inhibitors of LFA-1 are in clinical and preclinical
development for the treatment of inflammatory and immune diseases.
Aim: We studied the effect of two allosteric LFA-1 inhibitors with distinct
binding sites within the LFA-1 receptor on the receptor conformation
(1) , expression (2) and T-cell activation (3). A novel 4 color flow
cytometry method was developed to study these 3 parameters
simultaneously on single CD3+ T cells in whole human blood. Methods/
Results: The á/L L-site inhibitor LFA878 and the á/L I-like domain
inhibitor XVA143 potently induced distinct conformational changes
within LFA-1 receptors expressed on T-lymphocytes in human whole
blood. These nM activities became evident by assessing the binding of
conformation sensitive anti LFA-1 antibodies R7.1 (CD11a) and MEM48
(CD18) to whole blood T-cells in the presence or absence of the
inhibitors. Furthermore, XVA143, but not LFA878 significantly downregulated LFA-1 surface expression on CD3 + T-lymphocytes revealing
a novel property of á/L I-like domain inhibitors. XVA143 blocked anti
CD3 induced upregulation of the activation marker CD69 (IC50: 0.05
ìM) with higher potency than the clinically used immunosuppressant
cyclosporin A (IC50: 0.64 ìM). LFA878 blocked T-cell activation less
potently (IC50:1-2 ìM) than the á/L I-like domain inhibitor.
Conclusion: The established methodology enables to comprehensively
study pharmacodynamic effects of different classes of allosteric LFA-1
inhibitors in whole blood samples on individual T cells. Our results
suggest for the first time that distinct modes of allosteric LFA-1 inhibition
translate into differential and potent effects on the receptor level and the
immunologic function of LFA-1. Flow cytometry has proven an
indispensable technology to study these immunomodulatory drug
candidates in preclinical and potentially clinical investigations.
309/P163
SILICA - RED CELL ADSORPTIVE INTERACTION BY
LIGHT SCATTERING MEASUREMENTS
Igor I. Gerashchenko1, Bogdan I. Gerashchenko2, Vasyl F.
Gorchev3
1
Bogomolets National Medical University, Kiev, Ukraine;
University of Medicine and Dentistry of New Jersey, Pediatrics,
New Jersey Medical School, Newark, New Jersey; 3Palladin
Institute of Biochemistry, Kiev, Ukraine
2
As a result of adsorptive interaction between membranophilic silica
particles (silicon dioxide; SiO2) and red blood cells (RBCs), RBCs change
their shape and size, and can even be damaged, causing hemolysis. A
recent flow cytometry (FCM) based study of the light scattering
properties of RBCs interacting with SiO2 particles has demonstrated that
RBCs are capable of detecting changes in the surface properties of
particles ( Cytometry 2002;49:56-61). The current work describes a
study of silica - cell interaction by monitoring the changes in the size
distributions of interacting RBCs and SiO 2 particles. Washed human
RBCs interacted with SiO 2 particles of a 0.01% colloidal solution of
fumed silica (Aerosil A-300). Photon correlation spectroscopy (PCS)
capable of sizing the objects in mono- and polydispese systems was
used for characterizing the size changes of RBCs and SiO2 particles over
30 min of interaction. FCM and photometric measurements of released
hemoglobin were carried out to support PCS observations. During this
period of time, PCS revealed drastic changes in the size distribution of
RBCs as well as SiO2 particles (Fig. 1). These changes were expressed to
a greater extent during the rapid phase of interaction (i.e., first 10
minutes of interaction).
174
310/P164
MODULATION OF TOXICITY OF CYTOKINES
SECRETED FROM THE HUMAN MONOCYTIC THP-1
CELLS TOWARD SH-SY5Y NEUROBLASTOMA CELLS
BY SELECTED FLAVONOIDS AND THEIR
GLUCURONIDES
Jingli Zhang1, David Stevenson1, Aselle Adaim1, Roger
Stanley1, Margot Skinner1
1
HortResearch, Mt Albert, Auckland, New Zealand
INTRODUCTION: A sequentially-linked two cell culture system was
used to investigate the neurotoxic features of microglia. The THP-1
human monocytic cell line was used as an in vitro model of microglia.
The effects of supernatants from stimulated THP-1 cells on human
neuroblastoma SH-SY5Y cells were used as a model for glial cell
neurotoxicity and cytokines possibly associated with this neurotoxicity
were measured by using bead array technology. The anti-inflammatory
and neuroprotective effects of four flavonoids and their glucuronides
were investigated. METHODS: THP-1 cells were stimulated with LPS/
IFN-ã with and without the addition of test compounds. After 24h
incubation, cells were analysed for cell death by stained with AnnexinV-FITC and propidium iodide. An aliquot of cell-free supernatant was
analysed for cytokines by using a FlowCytomix kit, while another aliquot
was transferred to SH-SY5Y cells. Subsequently, SH-SY5Y cells were
incubated for a further 24h, harvested and subjected to cell death
analysis. RESULTS: Supernatants from stimulated THP-1 cells caused
the death of SH-SY5Y cells demonstrating neurotoxic effects. Pretreatment of the stimulated THP-1 cells with either flavonoids or their
glucuronides inhibited this toxicity. Similarly addition of the flavanoids
or their glucuronides directly to the SH-SY5Y cells inhibited the toxic
action from the stimulated monocytes. Control experiments showed
that the observed increase of neuronal cell death was due to substances
secreted from the stimulated THP-1 cells. All four flavonoids inhibited
the production of cytokines by THP-1 cells. Their glucuronides were
similarly active but those of phloretin and phloridzin were better at
inhibiting IL-6 whereas the glucuronides of catechin and quercetin were
better at inhibiting IL-1â, TNF-á and IL-12p70. There was little effect
of either type of compound on the production of anti-inflammatory
cytokines such as IL-10 and IL-8. CONCLUSIONS: By modulating
cytokine production, flavonoids and their derivatives could protect
neurons from glial cell induced toxicity. Here we describe a robust
model system based on the THP-1 and the SH-SY5Y cells, which may
reflect neuroneal-glial cell interactions. We provide evidence that a
group of flavanoids and their glucuronides, which are the more likely
form in which these flavanoids present themselves to cells in vivo, can
inhibit monocyte-induced neuronal death. These effects can result from
modulation of monocyte activation and inhibit the toxicity of factors
secreted from stimulated monocytes for neuronal cells. The results
demonstrate the usefulness of this two cell model system in screening
for the neuroprotective effects of phytochemicals.
311/P165
SINGLE CELL SORTING IN THE DESIGN OF AURORA
ASSAYS: SELECTION OF CELL POOLS EXPRESSING
BETA-LACTAMASE UNDER TWO DIFFERENT
PROMOTERS
1
2
Michael Cunningham , Marianna Kapitskaya , Bohumil
Bednar3, Stefanie Kane3, Konstantin Petrukhin2
1
Merck and Company, Inc., Neuroscience, West Point,
Pennsylvania; 2Merck & Co. Inc, Neuroscience, Opthalmics
Research, West Point, Pennsylvania; 3Merck & Co. Inc,
Neuroscience, Pain Research, West Point, Pennsylvania
Modern drug discovery has been based on high-throughput screening
using whole cell assays. A prominent role has been assigned to the
reporter gene technology based on a beta-lactamase and the fluorogenic
substrate CCF2. Successful application of this technology requires
fluorescence activated cell sorting (FACS). We developed a live-cell,
transcription-based beta-lactamase reporter assay suitable for highthroughput screening for RNR agonists.
Retina-specific nuclear
receptor (RNR) is an orphan nuclear receptor expressed exclusively in
photoreceptor cells of the retina. RNR mutations are associated with
photoreceptor degeneration in humans, such as age-related macular
degeneration (AMD). Loss of vision in AMD relates to degeneration of
photoreceptor cells in the central portion of the retina called the macula.
We developed a homogenous cell-based resonance energy transfer assay
for identification of RNR agonists using beta-lactamase as the reporter
gene. Bacterial beta-lactamase reporter construct containing GAL4
DBD response elements was randomly integrated into the genome with
subsequent selection of responsive cell pools by FACS. The choice of
beta-lactamase as a reporter was based on its following advantages over
other reporter systems: (1) signal transduction can be measured at the
level of the single living cell, (2) in conjunction with FACS, this system
provides an opportunity to develop stable cells with different levels of
beta-lactamase expression and to select pools of cells with optimized
response to nuclear receptor regulation, (3) the reporter expression is
monitored by the change in fluorescence of beta-lactamase substrate
from green (non-expressing beta-lactamase) to blue (expressing betalactamase) and is measured as the ratio of fluorescence at 460nm and
538 nm. The ratiometric nature of the reporter measurement provides
intrinsic “normalization” of the assay and is critical for successful
miniaturization of the assay to a 3456-well format with decreased wellto-well and day-to-day fluctuations.
313/P167
GABA-B AGONIST, POSITIVE ALLOSTERIC
MODULATOR AND ANTAGONIST ASSAY
DEVELOPMENT
Christopher Donahue1, Nicole Bart2, Mei Cui3, Brad Evans4,
Justin Van Duine5, Aaron Clark6, Fu-Zon Chung7
1
Pfizer Global Research and Development, Ann Arbor, Michigan;
Lexicon Genetics Incorporated, Research and Development, The
Woodlands, Texas; 3Pfizer Global Research and Development,
Molecular Pharmacology, Ann Arbor, Michigan; 4Pfizer Global
Research and Development, Biostatistics, Ann Arbor, Michigan;
5
Pfizer Global Research and Development, Materials Management,
Ann Arbor, Michigan; 6Pfizer Global Research and Development,
Assay Technologies, Ann Arbor, Michigan; 7Pfizer Global Research
and Development, CNS-Psychotherapeutics, Ann Arbor, Michigan
2
g-Aminobutyric acid-B (GABA-B) receptors are broadly expressed in
the nervous system and have been implicated in a wide variety of
neurological and psychiatric disorders. The only GABA-B drug on the
market is the agonist baclofen (Lioresalâ) that is used to treat severe
spasticity of cerebral and spinal origin. GABA-B antagonists show
great therapeutic promise but their shortcomings (lack of brain
penetration or proconvulsive potential) prevented clinical development.
Metabotropic GABA-B receptors are unique amongst GPCRs in their
requirement for heterodimerization between two subunits, GABA-B1
and GABA-B2 , for functional expression. GABA-B receptors trigger
second messenger systems through the binding and activation of Gproteins. Hypo-activation of the GABA-B system was suggested to
underlie epilepsy, spasticity, anxiety, stress, sleep, depression, addiction
and pain. In contrast, schizophrenia was associated with the hyperactivation of the GABAergic system (1,2,3) To identify novel GABAB agonists, positive modulators, and antagonists, a functional based
High Throughput Screen is necessary. To pursue this target, both GABAB1 and GABA-B2 receptors have been cloned and co-expressed in
Gqi(3)5, b-lactamase containing CHO cells. Using this cell line, a
sensitive High Throughput Flipr-based functional assay has been
established and validated.
314/P168
LIMITS OF MINIATURIZATION IN HIGH-CONTENT
ANALYSIS: HOW BETTER DATA EXTRACTION CAN
REDUCE CELL NUMBER REQUIREMENT
Ilya Ravkin1
1
312/P166
GABA-B CELL LINE DEVELOPMENT
Christopher Donahue1, Mei Cui2, Fu-Zon Chung3
1
Pfizer Global Research and Development, Discovery Biomarkers
Group, Ann Arbor, Michigan; 2Pfizer Global Research and
Development, Molecular Pharmacology, Ann Arbor, Michigan;
3
Pfizer Global Research and Development, CNSPsychotherapeutics, Ann Arbor, Michigan
The GABA-B (g-Aminobutyric acid-B) receptor is a member of the
“family 3” G-protein coupled receptors, which also consists of
metabotropic glutamate receptors (mGluRs). The GABA-B receptor
has a structure that is characterized by 7 transmembrane spanning
domains, and a large extracellular N-terminal ligand binding domain.
GABA-B receptors modulate activity inwardly rectifying potassium
channels and high voltage activated calcium chcnnels. GABA-B
receptors are unique amongst GPCRs in their requirement for
heterodimerization between two subunits, GABA-B1 and GABA-B2,
for functional expression. A cell line had to be developed that contained
both GABA-B1 and GABA-B2 receptors. Both receptors were cloned
and co-expressed in Gqi5, containing CHO cells. The cell line was
analyzed and sorted using b-lactamase as a reporter. GABA-B receptor
activation triggers calcium signaling through the binding and activation
of G-proteins. Calcium signaling thus triggers b-lactamase expression.
Single cell clones were sorted and isolated using flow Cytometry based
on high b-lactamase expression. High responders expressing GABA-B
receptor clones were further tested and evaluated on the Flipr. Using an
isolated single-cell clone, a sensitive Flipr-based functional assay has
been established and validated.
Palo Alto, California
There is always a desire to miniaturize assays, including cell-based
assays analyzed by imaging. This becomes a necessity when the number
of available cells is limited as with primary cells, or by design as in
multiplexed cell arrays. Among the factors that limit miniaturization is
increased variability of data due to small number of cells. Different
image analysis algorithms have varying ability to extract, from images,
the most stable cell features characteristic of a given assay. The better an
algorithm does this, the more precise will be the resulting data and the
lower will be the required cell number.
315/P169
RAPID NON-INVASIVE ASSAYS FOR CELL GROWTH
AND INHIBITORY EFFECTS OF COMPOUNDS ON
PRIMARY CULTURES OF HUMAN UNLABELED CELLS
Marc Moeremans1, Kris Ver Donck1, Bieke Govaerts1, Luc
Bols1, Leen Geuens1, Johan Geysen1
1
MAIA SCIENTIFIC, Geel, Belgium
The interest in automated cell based assays has greatly increased because
of the development of high information content screens using fluorescent
techniques. The availability of molecular biology tools to mark in situ
all kind of proteins with a fluorescent tag has allowed studying their
role in complex biological phenomena, such as signal transduction
pathways. However there is still a substantial need for generic assays to
read additional endpoints in the drug development chain. Preferentially
these assays are developed as such that they can be directly integrated
into the existing functional screening protocols. We have developed
automated brightfield imaging methods to detect and count living
175
unlabelled cells using the MIAS ® -2 microscopy reader and newly
developed eaZYX ® imaging routines. Two essential features of the
method are: i) the use of autofocusing algorithms based on scale space
mathematics allowing to capture high quality images in a fast way; ii)
the use of algorithms allowing to analyze images independent of their
variable background. The methods proved to be useful to count cells in
suspension cultures and to determine the stage of confluence in cultures
of adherent cells. Image capturing took about 3 seconds per sample
resulting in a plate cycle time of less than 5 minutes for a 96 well plate.
This rapid non-invasive cell count and/or confluence application was
used to study the effects of drug candidates on cell growth and cell
death in primary cultures of living human epidermal keratinocytes. The
generic nature and non-invasive character of the assay allows for intraassay assessment of toxic effects, as well as of stimulatory/inhibitory
effects of compounds on cell growth in a wide variety of existing
functional cell based screens.
316/P170
A NEW HIGH THROUGHPUT PROTEOMIC APPROACH
FOR RAID PROTEIN INTERACTION NETWORK
IDENTIFICATION USING TWO HYBRID SYSTEMS AND
CELL SORTING
Weon Bae1, Jian Hong Zhou2, Hong Cai1
1
Los Alamos National Laboratory, Bioscience, Los Alamos, New
Mexico; 2Los Alamos National Laboratory, Los Alamos, New
Mexico
The completion of the Human Genome Project holds great promise for
understanding and treating human disease. For this potential to be
realized, however, it is of critical importance to identify the structure,
function, and interactions of the proteins produced by individual genes
and their roles in specific disease processes. Various technologies have
been adopted to screen for protein-protein interactions, such as
immunoprecipitation, large-scale 3D structural analysis, array
technology of proteins (so-called protein chips), and high-throughput
two-hybrid methodology along with mass spectroscopy. The goal of
current study is to develop a high-throughput screening technology for
protein-protein interactions through the combination of yeast two-hybrid
system and flow cytometry, which is expected to improve the speed by
several orders of magnitude. Our yeast two-hybrid system is composed
of a bait plasmid (pTY137), a prey plasmid (pTM114), and a yeast host
strain (ITH5). pTY137 contains the sequence encoding the binding
domain (BD) of the copper-inducible transcription factor Ace1, which
is used to make a fusion protein with a target (bait) protein. On the other
hand, pTM114 has a sequence for the activation domain (AD) of the
Ace1, which will be used to construct a prey library. The yeast strain
contains multiple copies of the copper resistance-mediating CUP1 (yeast
metallothionein) gene as well as the green fluorescent protein (GFP)
reporter gene stably integrated on its chromosome. A positive control
cells (ITH12), which express both a fusion protein of ACE1 binding
domain with N-terminal dimerization part of cAMP-dependent protein
kinase (ACE1 BD-BCY1) and ACE1-AD-BCY1, showed significant
fluorescence signal only upon the exposure to copper ions. All other
negative controls and a positive control didn´t produce fluorescence
signal above background level. We are currently under investigation to
screen protein-protein interactions using pre-selected bait proteins with
prey proteins constructed from cDNA library of Human blood cells.
317/P171
USE OF FLOW CYTOMETRY TO SELECT LEAD
THERAPEUTIC ANTIBODY CANDIDATES
Masahisa Handa
1
1
XOMA (US) LLC, Preclinical, Berkeley, California
Flow cytometry is a sensitive cellular analysis technology that can be
used to efficiently screen antibodies to select lead therapeutic candidates
for oncology and non-oncology indications. Using standard flow
cytometers equipped with microtiter plate acquisition and kinetic
stimulation injection capabilities (Cytek Development), hundreds of
antibody candidates targeting both cell surface and secreted molecules
can be screened. To exploit the flexibility, sensitivity and rich data
content of a FACS-based assay format and obviate the need for multiple
specialized HTS instruments, we routinely design customized testing
funnels to include various biochemical and/or biophysical assays for
binding selectivity and affinity upstream of cell-based assays. This
176
sufficiently narrows the number of candidates proceeding to the critical
flow-based functional screen. Examples of successful flow-based lead
selection campaigns involved targeting a GPCR ligand and a receptor
tyrosine kinase (RTK). For the GPCR ligand, a calcium-flux assay was
developed using the calcium indicator dyes Fluo-3 and Fura Red as the
primary functional assay to determine neutralization activity of the
anti-ligand antibodies. The assay was used for both primary screening
of antibody fragments generated from multiple human antibody phage
display libraries and for final lead selection using full IgGs. Prior to
testing in the calcium-flux assay, a relative off-rate ranking was
performed on the antibody fragments using Biacore(R) to narrow the
candidates tested to those with the highest affinity. The top unique
clones from the primary calcium-flux screen were reformatted to whole
IgG and tested in a 2-point assay relative to a mouse reference antibody
to further distinguish the top neutralizers. The lead mAb was selected
based on an IC50 determination of the greatest potency in addition to
affinity. For the RTK target, a total cellular phospho-tyrosine (pY) and
cell surface steady-state assays were developed as the primary functional
screens to determine agonistic activity of the anti-RTK Abs. These assays
were designed to assess receptor-mediated signaling and down-regulation
of cell surface receptors, respectively. Using the assays in a 96-well
format, a number of agonistic antibodies with similar or improved
potency compared to the natural ligand were identified.
Immunoprecipitation-Western analysis confirmed the agonistic Abs
identified by FACS induced receptor auto-phosphorylation. Western
analysis also was used to confirm total cellular degradation, again, a
finding consistent with the FACS-based assay. In summary, flow
cytometric analysis is a viable cost-effective screening platform to identify
rare functional lead therapeutic antibody candidates
318/P172
APPLICATION OF MICROPLATE CYTOMETRY TO HIGH
CONTENT SCREENING IN ONCOLOGY RESEARCH
Wayne P. Bowen1
1
TTP LabTech Ltd., Melbourn, Hertfordshire, United Kingdom
Oncology research has been quick to adopt High Content Analysis
(HCA) due to good compatibility between the biological applications
available and the screening requirements. Key applications include
protein kinase activation, protein translocation, cell cycle analysis and
cell colony formation. In addition, there is growing use of RNAi
technology for the identification and validation of oncology targets.
Traditional methods for HCA use technologies such as flow cytometry
and microscope-based imaging systems. Laser-scanning microplate
cytometry has many advantages over these, and is more amenable for
use in High Content Screening. For instance, it gives the ability to
analyse the entire well area of any SBS standard multiwell plate. This
enables identification of patchy cell distribution or cell stimulation by
compound. It also allows the user to analyse live or fixed cell assays in
both adherent and non-adherent cell lines. In addition to these advantages,
microplate cytometry offers rapid read and analysis times of plates
(typically 4 - 10 minutes) and produces small file sizes, therefore removing
the requirement for expensive data storage and retrieval solutions for
compound screening initiatives. This poster will provide an overview
of the main uses of an Acumen Explorer microplate cytometer in
oncology research.
319/P173
CELL-BASED IMAGING TOOLS FOR HIGH CONTENT
SCREENING
Oleg Lapets1, Everett Ramer1, Richik N. Ghosh1, Jeffrey R.
Haskins1
1
Cellomics, Inc, Pittsburgh, Pennsylvania
High Content Screening (HCS), based on automated extraction of data
from cellular images, is an indispensable screening and investigative
tool for drug discovery and development. HCS requires image
processing and analysis algorithms that can accurately and robustly
analyze large image numbers without requiring human intervention.
Most cell biological problems to be analyzed using HCS can be
categorized by either the changes in cell phenotype or labeling pattern.
This enables the application of directed algorithms optimized for these
categories. A suite of algorithms that are guided by an understanding of
the biology being studied was developed for the optimized automated
acquisition, quantitation and analysis of cellular and sub-cellular
structures. Two categories of these directed algorithms were developed:
General Purpose Algorithms, tools for assay development, and Specific
Algorithms which are turnkey solutions for specific biological screening
assays. These algorithms comprise a well established sequence of image
processing and analysis steps that include: primary object identification;
measurement of primary object properties; identification and
measurements of associated targets; and analysis of raw measurements
for specific biological applications. Examples of practical utilization of
these algorithms in typical HSC applications are presented and discussed.
320/P174
AUTOMATED MICROINJECTION SYSTEM FOR
SUSPENDED AND ADHERENT CELLS
Ian Welsford1
1
BioSciences Group, Fujitsu Computer Systems, Westwood,
Massachusetts
Transfection in multipotent and progenitor cells, including human
embryonic stem cells (ES), is hindered by the fact that such cells are
frequently refractory to lenti- and retroviral-based vectors. We report
preliminary tests using a system designed to automate the microinjection
process for such refractory cells. The system is capable of handling
both suspended and adherent cells. For the suspended mode of operation,
a 3.5 mm2 silicon chip was perforated with holes. To measure the apex
position of the capillary, a red light, which transmits through the chip,
is illuminated from above and detected by a CCD camera set on an
inverted microscope. When the hole positions are detected, the
illuminating light is switched to a green light. The green light is strongly
absorbed by the silicon chip, and thus the light is transmitted only
through the holes. From analyzing the captured images, positions of the
capillary as well as well holes are determined precisely by computer
control. When medium containing cells is placed on the chip and negative
pressure is applied from underneath, cells are captured at the upper
openings of the holes. The chip can hold up to 1043 cells simultaneously.
In the adherent mode, tissue culture dishes are directly loaded onto the
device and cells are injected in situ. The system records the cells in each
area of the dish that have been injected to facilitate downstream
processing. In the adherent mode, the system has an average throughput
of 500-1000 cells per hour, depending upon culture density. To test the
efficacy of the system on various cell types, we injected K562 cells
(suspended mode), P388D1 cells (suspended mode), IC-21 cells (adherent
mode), NIH/3T3 cells (adherent mode) and HL60 cells (suspended
mode). For each experiment, cells were injected with a capillary filled
with 2 mg/mL QDot 655 antibody conjugate (655 nm) and 0.5 Ýg/ÝL
GFP vector pQBI-B23GFP . Red fluorescence was determined 15 minutes
after injection to determine injection rate while GFP signal was
determined 24 and 48 h after injection to determine expression efficiency.
Injection efficiencies for K562, P388D1, IC-21, NIH/3T3 and HL-60
cells averaged 68%, 75%, 99%, 93% and 59% respectively while
expression efficiencies averaged 98%,76%, 74%, 81% and 62%
respectively. Work is ongoing to determine the efficiency of handling
human ES.
321/P175
HIGH-THROUGHPUT SCANNING CYTOMETRY WITH
LASER OPTO-INJECTION OF MACROMOLECULES
INTO SELECTED CELLS (WITH LASER-MEDIATED
ELIMINATION OF UNDESIRED CELLS) FOR
SUBSEQUENT GENE ARRAY ANALYSES
James F. Leary1, Peter Szaniszlo2
1
Engineering, West Lafayette, Indiana; 2University of Texas Medical
Branch at Galveston, Microbiology & Immunology, Medicine,
Galveston, Texas
Scanning cytometry has many of the features multiparameter flow
cytometry while keeping its own advantages as an imaging technology.
A new imaging instrument, called LEAP™ (Laser Enabled Analysis
and Processing)(Cyntellect, Inc. San Diego, CA), offers a unique
combination of capabilities in high-throughput imaging, cell purification,
and selective macromolecule delivery (optoinjection).
LEAP-mediated
cell purification and optoinjection effects were assessed in model
experiments using both adherent and suspension cell types and cell
mixtures plated and processed at different densities. Optoinjection effects
were visualized by delivering fluorescent dextrans into cells. These can
be co-optoinjected with other molecules such as small gene sequences
and GFP reporter genes, siRNAs, and QDot(TM) nanocrystals. Results
were analyzed using the LEAP instrument´s own imaging system as
well as by fluorescence and confocal microscopy. Non-opto-injected
cells can be eliminated from the cell mixtures using the laser ablation
mode of LEAP at higher energies than the optoinjection laser power
densities.
Live cell samples (both adherent and suspension) were
purified to 90-100% purity with 50-90% yield, causing minimal cell
damage depending on the cell type and plating density. 100% of the
targeted cells of all cell types examined were successfully optoinjected
with dextrans of 3-70kD, causing no visual damage to the cells. Indirect
optoinjection effects were observed on untargeted cells within 5-60µm
to targeted areas. LEAP has minimal effects on gene expression profiles
(GEP) of optoinjected cells using Affymetrix gene arrays. Likewise, the
GEPs of cells recovered after ablating undesired cells appear similar to
those of the original pure cell type that has not been subjected to LEAP
analyses.
LEAP technology provides many of the advantages of LCM
and laser catapulting, as well as the ability to manipulate single cells by
a variation of laser tweezer technology. LEAP provides a method of
live cell sorting based on laser ablation. Additionally, the LEAP
instrument provides a convenient method for high-speed microinjection
of macromolecules into living cells using a pulsed laser set at sub-lethal
energies. This optoinjection process is fast (hundreds of cells/sec) and,
unlike electroporation, has a very low rate of injury to cells which can
be individually selected on the basis of multiple fluorescent probes in
an automated molecular imaging process. LEAP can be performed in a
totally sealed environment in a process akin to inverted fluorescence
microscopy with long-working length distance objectives. It also offers
a new method of sorting or optoinjecting cells under sterile conditions
and provides a safe means of handling biohazardous cells and agents.
322/P176
LASER OPTOINJECTION OF THERAPEUTIC
NANOPARTICLES INTO OSTEOSARCOMA CELLS
Michael D Zordan1, Lisa M Reece2, James F. Leary2
1
Lafayette, Indiana
Osteosarcoma is a prevalent form of cancer that affects mainly juvenile
subjects. It is characterized by large tumors that develop in the growth
plate of long bones. Current treatment options are chemotherapy
supported amputation or limb salvaging surgery where an autologous
bone graft fills the void created by the removal of the tumor. Because
of these extreme treatment options, osteosarcoma is a prime candidate
for the development of regenerative gene therapy.
Possible
regenerative therapy is being explored in a new project using LEAP™
(Laser-Enabled Analysis and Processing)(Cyntellect, Inc.) technology.
LEAP™ technology combines the imaging and analysis of scanning
cytometry with the ability to purify cells using laser ablation and insert
macromolecules into cells using optoinjection. LEAP™ can be used in
conjunction with gene micro-arrays to determine the genomic differences
between healthy and malignant osteoblasts.
Gene therapy and
wound healing agents will be delivered to cells in the affected regions
using nanoparticles containing therapeutic genes or growth stimulating
peptides. The agents can then be released by laser-mediated heating.
LEAP™ will be used to target malignant cells fluorescently tagged with
biomarkers or morphologically distinct. Once cells have been targeted,
the nanoparticle will be delivered into the cells using optoinjection.
The layered structure of the optoinjected nanoparticle will then
sequentially target the cells nucleus and specific genes. The gene therapy
agent will then be released by melting away the outer shell of the
nanoparticles. This melting can be performed ex-vivo by specifically
microheating the target cell nucleus using the laser of the LEAP™
system. In-vivo heating can be accomplished with ultrasound
stimulation.
Three dimensional tissue culture models of both rat and
canine osteosarcoma are being used to develop this regenerative therapy.
An example of eventual clinical use would be to use this therapy to
regenerate healthy tissue from excised tumors and use this as an
autologous bone graft as opposed to the harvested pelvic bone that is
currently used.
177
323/P177
AUTOMATING LARGE SCALE, REPETITIVE, CLINICAL
FLOW CYTOMETRY ANALYSIS WITH THE
CUSTOMIZED PROTOCOL ENGINE FLOWDX
Adam Treister1
1
Tree Star, Inc., Ashland, Oregon
This poster illustrates the characteristics of an FCS data analysis tool
optimized to inexpensively perform complex operations with minimal
user input. Preconfigured per experiment, it can predict materials needs,
and produce test results in multiple media. It is comprehensive
experiment design software – allowing the scientist to design, schedule,
prepare and control experiments. The Protocol Editor provides a dragand-drop interface wherein the parameters of the study can be entered
and visualized. This information updates the cytometer´s worklist
manager so that each successive run of the procedure is accurately and
identically analyzed and displayed. The framework of the experimental
design can then be saved and reloaded independent of experimental
data so that future iterations of the same experiment will be displayed
and analyzed consistently without manual configuration of worklist or
software. This is of particular interest to the growing cadre of users in
the clinical laboratory where experiments are repeated many times and
where accuracy and consistency are of the utmost importance. FlowDx
will supply customized protocols and tools to both the research and the
clinical arm of cytometry. It will present a specialized interface to the
operator based upon the demands of each experiment.
324/P178
USING THE POLYVARIATE PLOT TOOL
Adam Treister1, Maciej Simm1
1
Tree Star, Inc., Ashland, Oregon
This poster describes a new graph type and gating tool that displays
multiple parameters in two dimensions. Creating gates on the PolyVariate
Plot shortcuts the repetitive processes of hierarchical and boolean gating.
The display updates in real time and many parameters can be isolated,
displayed and gated at once. This tool presents a new perspective on
multicolor FCS data. Multiple parameters are entered as user adjustable
vectors projected onto a polar co-ordinate display. Each vector imparts
a magnitude and a direction to its parameter. An event´s position is
determined by the combination of its vector values. By manipulating
the display´s variables the user can move subpopulations of interest into
distinct clusters for quick gating.
325/P179
SCREENING FOR PLANT BIOCATALYSTS USING
REACTIVITY BASED PROBES
Aileen Congreve1
1
University of Durham, Department of Chemistry, Department of
Chemistry, Durham, England, United Kingdom
Potential advantages of biotransformations in preparative and
bioremediation applications have been acknowledged for decades. In
this context interest lies particularly in plant systems which represent a
particularly diverse and largely unexploited range of enzymes. Most
current biocatalysis screening strategies have relied on the expression
of the recombinant enzymes in bacterial hosts. Such an approach has
limitations if the cellular environment required to support catalysis is
absent in the microbial host. Recent advances in plant transformation
technology potentially allow us to exploit plant cells as a generic host
for biocatalyst transgene expression. In addition to overcoming the
limitations of microbial hosts systems, this relatively unexplored strategy
permits the exploitation of the far greater natural diversity of biocatalysts
found in homologous plant based systems. This presentation will
describe the development of smart pro-fluorescent probes which are
178
selectively bio-activated allowing the identification and isolation of the
enzyme of interest by flow cytometric sorting of large transgenic
protoplast populations.
326/P180
HIGH THROUGHPUT METHOD FOR THE
IDENTIFICATION OF IN VIVO PROTEIN
INTERACTIONS AND MODIFICATIONS DURING D.
MELANOGASTER EMBRYOGENESIS
Joshua Wunderlich1, Erika Geisbrecht1, Kiranmai
Kocherlakota1, David Ash1, Mei-Hui Chen1, Selene
Swanson1, Laurence Florens1, Michael Washburn1, Susan
Abmayr1, Jeffrey Haug1
1
Stowers Institute for Medical Research, Kansas City, Missouri
Drosophila melanogaster is an ideal model organism for the study of
developmental processes. Past and ongoing research has demonstrated
its use in classical genetics where the study of mutations and their resulting
phenotypes have been characterized. However, this model system has
not proven ideal for studies addressing biologically relevant protein
interactions in the Drosophila embryo, as it is difficult to obtain quantities
of protein sufficient for analysis. With current methods, it is possible to
direct expression of a tagged protein of interest to particular tissues in
the fly, and to determine whether this tagged protein is functional.
However, it has remained an intractable problem to obtain sufficient
material for purification of the protein and its subsequent analysis by
mass spectrometry. One main hindrance lies in the effort needed to
collect virgin females for large scale production of tagged protein.
Using a COPAS Plus macro particle sorter, we have been able to purify
large populations of transgenic female embryos and synchronize their
arrival into mating age. With 6 hours of sorting, we can obtain over
35,000 viable females embryos. The resulting adult females, which
express a muscle-specific driver, are then crossed to transgenic males
expressing an affinity-tagged protein of interest. The embryos produced
by these adults are then collected for lysis, and the tagged protein, in
turn, is purified by immunoprecipitation. From this procedure, the
purified product (and any interacting proteins) can be submitted for
shotgun proteomics analysis. Such a method has proven to be a powerful
tool for in vivo proteomic studies of myogenesis. Approximately 300
ìg of embryonic lysate is required to obtain enough immunoprecipitated
material for one mass-spectrometry sample. In general, enough lysate
is obtained from the above sort for preparation of approximately 3-5
samples allowing for replicate analyses. Therefore, through COPAS
sorting, we have shown that we can substantially decrease the amount
of time required for virgining and collect sufficient quantities of lysate
for mass spectrometric analysis, making such analyses feasible. With
the use of the aforementioned sorting process and proteomics analysis,
we were able to identify our tagged protein as well as anticipated
interacting proteins. We have also identified novel protein associations
and putative protein modification events that are being validated. It is
our belief that this principle can be applied to the study of the development
of many other tissue types during embryogenesis, and potentially even
other small, multicellular organisms, such as zebrafish and C. elegans.
327/P181
EVALUATION OF A SINGLE AUTOMATED PLATFORM
FOR THE STANDARDIZATION OF FUNCTIONAL CELL
BASED ASSAYS IN PLATES
Julie G. Wilkinson1, Aaron Globerman1, Carlos Luis
Aparicio1, Sybil S. D’Costa1, Cecilia Smith1, Enrique
Rabellino1
1
Beckman Coulter Inc., Custom Biopharma Solutions, Miami,
Florida
The use of cell-based assays in high throughput drug screening as well
as biologic development in both research and clinical trial settings has
grown due to their relevance to in vivo settings. Although there are an
increasing number and diversity of functional cellular assays, the utility
of these assays has not been fully recognized due to their highly variable
nature. The variability arises from a variety of factors ranging from
source of the cells, kind of assay, sample processing methodology
(isolation, freezing, thawing, and culturing), sample staining protocol
and ultimately the bioinformatics. With a view to standardizing the
preparation of functional cellular assays in plates, the Biomek3000
automated workstation was evaluated. Untreated peripheral blood
mononuclear cells (PBMCs), were fixed and permeabilized on the Biomek
3000 using a validated protocol for intracellular cytokine analysis. The
versatility of the platform enabled the integration of the MultiScreen
HTS Filter Manifold for non-centrifugal washing and recovery of cells
in suspension. To optimize the wash protocol, combinations of many
filtration plate formats from Millipore (membrane type, pore size) and
mixing mechanisms to recover cells from the filter were evaluated also.
The optimum conditions for the assay included the use of PVDF filter
membranes with a 0.45mM pore size. The assay enabled cell recoveries
of >80% without selective cell loss. The precise pipetting, timing, and
washing of cells in suspension on a single platform, in the absence of
centrifugation, greatly improved sample processing uniformity and
reduced the variability inherent to functional assays as compared to the
manually performed procedure. Additionally, the automation enabled a
significant reduction of “hands-on” sample preparation and analysis
time. The use of automation thus provides a greater degree of
standardization in these functional assays, allowing for their use in
applied research settings as surrogate markers of efficacy or activity.
329/P183
THE PLACE AND USE OF AN IMAGE SCANNING
CYTOMETER IN A FLOW CYTOMETRY CORE
Gregory H. Veltri1
1
University of Minnesota, Cancer Center Research Building,
Minneapolis, Minnesota
University of Minnesota Cancer Center flow cytometry core facility is
not unlike any other flow core. We strive to be essential component of
our research institution and to provide the best technology for our
users. We offer training, assistance and perform flow cytometry
techniques and experiments to all the members of the Cancer Center.
Our core´s users have often needed to visualize their cells and determine
exactly what is occurring with the cells. In many instances our users use
a confocal microscope, but doing so sacrifices the number of cells that
can be observed as well as fluorescent quantification. For these reasons
we purchased and began using the Image Stream 100 from Amnis Corp.
The Image Stream´s ability to take a picture of each cell that passes
through is extremely valuable for a flow cytometry core. The core and
its users are given a tool that allows them to visualize the location of
their fluorescent markers, examine many cells, and quantify the data
collected. In conclusion, the flow core facility now has a tool for our
users to find interesting answers to some old questions by using the
Image Stream.
330/P184
LASER SCANNING ANALYSIS OF CHROMATICALLY
LABELED TISSUE SECTIONS
Ed Luther1, Lori Earls2, William Geddie3
1
CompuCyte Corporation, Cambridge, Massachusetts; 2Brody
School of Medecine, Pathology, Greenville, North Carolina;
3
Quantitative analysis of chromatically stained tissue sections by laser
scanning light absorption measurements is being applied in many areas
of clinical and biomedical research. We describe two models we have
developed for analysis of paraffin sections labeled by
immunohistochemical methods: one using two-color analysis and the
second using a more sophisticated three-color analysis. The two-color
model is a human lymph node labeled with DAB-developed Ki67 (clone
MIB1) and counterstained with hematoxylin. The section was scanned
on an iCyte® Automated Imaging Cytometer. An initial high-speed
scan generated a mosaic image, from which regions of interest were set
for high-resolution scans. Laser light absorption from 488nm and 633nm
lasers was measured with photodiode detectors, while autofluorescence
was measured with photomultiplier tubes. Absorption signals were
corrected for spectral overlap, so that “virtual channels” of specific
Ki67 staining and hematoxylin staining were obtained. The spectral
compensation algorithms were effective, and overlap of the DAB was
completely eliminated from the hematoxylin images. DNA density
differences were clearly shown in the germinal centers, indicating
quantitative DNA density measurements. Nuclear-based segmentation
(including watershed algorithms to separate closely spaced nuclei) and
random segmentation strategies gave similar results over a large range
of the proliferation indices (MIB1 labeling) and across different portions
of the tissue. The correlation between the two segmentation methods
yielded an R2 value of 0.986. The second model is a three-color
system. A renal carcinoma section stained with CAIX (DAB) and CD34
(Vector Red) and counterstained with hematoxylin was analyzed on an
iColor™ Fluoro-chromatic Imaging Cytometer with red, green and
blue simultaneous laser excitations and separate detectors. Spectral
overlap compensation successfully separated all three staining
constituents, and the inherent fluorescence of Vector Red permitted
secondary verification of the absorbance correction algorithm with this
chromogen. The method has been validated with other multiple-dye
combinations. The results show that LSC technology, previously
employed for fluorescence-based tissue analysis, is able to resolve and
quantify multiple chromatic labels in immunohistochemical preparations.
This capability has important implications in histological and pathological
analysis of tissue sections, both as they are related to biomarker
development and, clinically, for multiplexed tumor marker studies in
limited diagnostic samples.
331/P185
DIFFUSION PROPERTIES OF LIPOPHILIC DYES
WITHIN AND BETWEEN ADJACENT CELL
MEMBRANES
Bernd Fritzsch1, Katharine A. Muirhead2, Brian D. Gray3,
Feng Feng1, Betsy Ohlsson-Wilhelm4
1
Creighton University, Department of Biomedical Sciences, Omaha,
Nebraska; 2SciGro, Inc./MidWest Office, Madison, Wisconsin; 3PTI
Research, Inc., Exton, Pennsylvania; 4SciGro, Inc./NorthEast
Office, Cambridge, Massachusetts
Introduction: Lipophilic dyes have been extensively used for neuronal
tracing n formalin fixed tissue, but their use for multicolor studies have
been limited by poorly matched diffusional and/or spectral properties.
A new family of probes, the NeuroVue™ dyes, combines well matched
diffusional properties with spectral properties optimized for use in laser
confocal microscopy ((Fritzsch et al., Brain Res Bull 2005). While
studying the diffusional properties of the next generation dye candidates
vs. DiI, the standard in the field for 20 years, we unexpectedly found
that dye transfer could be used to highlight cell-cell connections in
fixed tissue. Methods: Accurate dye diffusion comparisons in fixed
embryonic tissue must address issues such as precision of dye appli

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