The path to new discoveries

Transcription

Vol 15 – 1/2013
The path to
new discoveries
Highly reliable gene
expression profiling
Isolation of B cells by
MACSxpress® Technology for
sensitive and concordant
microarray analysis (p. 10)
Multiple myeloma
research
Automated purification of
CD138+ cells from whole bone
marrow for FISH analysis (p. 18)
Malaria research
Rapid, automated purification
of Plasmodium falciparum–
infected erythrocytes for
biochemical studies (p. 31)
From basic to clinical
research in immunology
Contents
Page
News
Enjoy our redesigned website 4
Meet our best kits yet
5
As fast as it gets: cell isolation from whole blood with MACSxpress® Technology
6
Automated workflows in immunology research – using simplicity to control complexity
7
The CliniMACS® CD34 Reagent System – a landmark in cellular therapy
8
Reports
MACSxpress® Technology allows isolation of B cells with high purity
for sensitive and concordant microarray-based gene expression profiling
Angela Mekes, Stephanie Soltenborn, Ines Dischinger, Volker Huppert,
and Bernhard Gerstmayer
10
Isolation of monocytes with high purity directly from whole blood for transcriptome analysis in translational research
Christelle Foucher, Sébastien Vachenc, Alexandre Meniccaci,
Bernhard Gerstmayer, Gwenola Henrion, Uwe Janssen,
Darren Wilbraham, Karine Le Malicot, and Jean-Claude Ansquer
14
An automated method for purification of CD138+ cells from whole bone marrow samples for multiple myeloma research
Hossain Mossafa and Sabine Defasque
18
Highly purified peripheral blood γ/δ T cells isolated by MACS® Technology
respond to NOD2 ligand
Lothar Marischen, Hans-Heinrich Oberg, Christian Peters,
Sandra Ussat, Hoa Ly, Dieter Kabelitz, and Daniela Wesch
20
Efficient and rapid in vitro generation of fully functional
multi-virus-specific CD4+ and CD8+ T cells
Anna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer,
Jürgen Schmitz, Mario Assenmacher, and Anne Richter
24
Mouse NK cells isolated to high purity
by MACS® Technology are fully functional
Kathrin Meinhardt, Irena Kroeger, Sabine Mueller, and Evelyn Ullrich
28
Measuring prolyl aminopeptidase activity in extracts prepared from
magnetically purified malaria parasites
Fabio L. da Silva, Donald L. Gardiner, and Katharine R. Trenholme
31
MACS&more (ISSN 1610-4994) is published by
Miltenyi Biotec GmbH.
Editorial board: Anthony Allen, Adrian Arechiga, Conrad Beckers,
Caroline Blumer Toti, Sven Brosch, Oliver Burlon, Vincent Chu,
Steven Dublin, Guy Hewlett, Christoph Hintzen, Jennifer Horner,
Volker Huppert, Claudia Loske, Joumana Masri, Simon Mauch,
Mariette Mohaupt, Shane Oram, Gerd Steffens, Nanette von Oppen,
Ilka Wege, Raif Yücel
Editor: Ralph Schaloske
Graphics & Layout: Miltenyi Biotec GmbH, Graphics team
Miltenyi Biotec GmbH
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Unless otherwise specifically indicated, Miltenyi Biotec products
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purposes only.
Copyright © 2013 Miltenyi Biotec GmbH. All rights reserved.
2 MACS & more
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• 1/2013
Dear Researcher,
Detailed research on the properties and
function of individual immune cell types
deepens our understanding of their role in
health and disease. With more than twenty
years of experience in biomedical research,
Miltenyi Biotec provides a multitude of
sophisticated tools for immune cell isolation
– helping advance research towards the
development of future therapies.
Usually, the immune system effectively
protects the body against disease. However,
there are myriads of scenarios in which the
immune system is not able to cope with
certain challenges, for example, the escape
of tumor cells, prolonged inflammation, or
autoimmune reactions, all of which can lead
to debilitating disease. The immune system is
based on a highly organized interplay between
a multitude of different cell types each
playing a distinct role. In order to accurately
analyze the function and characteristics
of a specific immune cell type in health
and disease, it is essential to use a pure cell
population. Miltenyi Biotec offers the tools
to isolate immune cells and subsets reliably
and efficiently. In this MACS&more issue
we present a selection of reports featuring
our solutions for magnetic cell separation
in a broad spectrum of immunology-related
applications.
lipid-lowering drug fenofibrate. The analysis
of differentially expressed genes enabled the
construction of functional networks.
For their research into malignant cells
from multiple myeloma, Hossain Mossafa
and Sabine Defasque required a reliable,
fast, and standardized technique to enrich
CD138+ plasma cells from a large number
of bone marrow samples. The autoMACS
Pro Separator and Whole Blood CD138
MicroBeads met all these requirements.
Using the isolated CD138+ cells for FISH
analysis, the authors were able to reliably
detect chromosomal abnormalities that are
characteristic for malignant plasma cells.
Lothar Marischen et al. investigated the
function of the innate receptor NOD2
expressed on γ/δ T cells. For their studies, the
authors needed γ/δ T cells and monocytes
with an exceptionally high level of purity.
MACS® Technology enabled Marischen et
al. to isolate both cell types from PBMCs to
purities of greater than 99%. Likewise, they
depleted either cell type from PBMCs to
residual percentages of less than 0.1%. Their
results support the notion that γ/δ T cells play
a role in anti-bacterial immunity.
spleen turned out to be challenging. As the
C57BL/6 strain is an important model in preclinical NK cell research, Meinhardt et al. set
out to solve this issue. They further optimized
the depletion procedure and developed a new
depletion cocktail, which is now part of the
NK Cell Isolation Kit II. This kit enables the
isolation of NK cells to high purities from
both BALB/c and C57BL/6 spleens. The
report by Meinhardt et al. is a great example
of how our collaboration with scientists using
Miltenyi Biotec products can lead to the
development of exciting new research tools.
In their quest to identify new targets for
antimalarial drug development, Fabio da Silva
et al. required preparations of Plasmodium
falciparum-infected red blood cells (RBCs)
with high purities. To this end, the authors
magnetically enriched infected RBCs
using the autoMACS Pro Separator. Unlike
immunomagnetic cell isolation based on
MACS Technology, their approach did not
involve a magnetic labeling step. Instead
the parasites labeled themselves: During
development P. falciparum digests the
host cell’s hemoglobin. This leads to the
formation of insoluble hemozoin, which has
paramagnetic properties. Da Silva et al. were
able to identify prolyl aminopeptidase activity
in extracts prepared from the magnetically
enriched parasites – a first step towards the
identification of a potential target for drug
development.
The reports by Angela Mekes et al. and
Christelle Foucher et al. highlight the
intriguing benefits of our tools for cell
isolation directly from whole blood for
subsequent microarray-based analyses. Using
the new MACSxpress® Technology, Mekes
et al. showed that B cells isolated from
whole blood enabled a more conclusive
analysis of cell type–specific gene expression
than unseparated whole blood or PBMCs.
MACSxpress Technology thus provides an
excellent basis for biomarker research.
Adoptive transfer of antigen-specific T cells
holds great potential for the control of
infections after stem cell transplantations.
To enhance research into virus-specific T cells,
Anna Foerster-Marniok et al. developed
a fast and straightforward protocol for
the generation and enrichment of T cell
populations that are specific for three different
viruses. The authors used Miltenyi Biotec’s
PepTivator® Peptide Pools for cell stimulation
and the MACS® Cytokine Secretion Assay
technology to isolate the virus-specific T cells.
Isolated virus-specific T cells were fully
functional and could be expanded easily.
Foucher et al. used the autoMACS® Pro
Separator in combination with Whole Blood
CD14 MicroBeads for the fast and reliable
isolation of monocytes. The authors analyzed
the transcriptome of monocytes purified
from whole blood obtained from healthy
donors before and after treatment with the
For their studies on the function of NK
cells, Kathrin Meinhardt et al. obtained
highly purified cells from BALB/c mouse
spleen using two strategies based on MACS
Technology: positive selection and depletion
of non-NK cells. However, the isolation of
high-purity NK
cells from
C57BL/6 mouse
MACS&more
online:
www.miltenyibiotec.com/macs&more
We wish you an inspiring read.
Your MACS&more team
Vol 15 • 1/2013
MACS & more 3
NEWS
Enjoy our redesigned website
With you, the busy scientist, in mind, we have redesigned our website.
Various new features make miltenyibiotec.com faster, easier, and more intuitive.
Save time with easy navigation
The simple interface and straightforward
navigation guides you to the information you
need in no time.
The revamped miltenyibiotec.com offers you
in-depth insight into products and services
for research and its translation into clinical
applications. You will also find comprehensive
information on individual research areas and
numerous support options, as well as general
company information.
Easy navigation is key to finding appropriate information fast.
Start your immunology experiment here
To keep you up to date on our wide range
of products for your immunology research,
we offer a special section on immunologyrelated topics, including autoimmunity,
tumor immunology, dermatology, allergy,
and infection and inflammation. Discover a
plethora of smart products for your sample
preparation, cell separation, flow cytometry,
molecular applications, and pre-clinical
imaging. Also benefit from numerous
references and downloads.
Find the products you need in just a
few clicks
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Welcome to the immunologist’s resource.
4 MACS & more
Vol 15 • 1/2013
Start enjoying the new features today at
NEWS
Meet our best kits yet
Miltenyi Biotec introduces the next generation of immune cell isolation kits. Exciting new updates have been made to some of our most
popular kits, resulting in new kits that offer unmatched purity, greater recovery, and faster cell isolation.
Miltenyi Biotec’s continuous quest for product labeling, and in many cases it became possible
refinement has led to the next generation of to omit washing steps. Mouse NK cells, for
kits for the isolation of plasmacytoid dendritic example, can now be isolated in less time
cells (PDCs), monocytes, NK cells, pan T cells, with unrivaled purity using the new NK Cell
and T cell subsets. Updates were made on Isolation Kit II. Pure pan monocytes, pan
various levels. Two kits for the isolation of T cells, as well as CD4+ and CD8+ T cells can
human PDCs were revamped with depletion be obtained in as little as 25 minutes and the
cocktails that were redeveloped from scratch, isolation of human Treg cells or NK cells can
allowing for unmatched PDC purity and be accomplished up to 20 minutes faster than
recovery. Regular communication with users before.
of our products allowed us to optimize the “The original kits were already very effective
kits according to the users’ specific needs. The and immensely popular. However, when we
Pan Monocyte Isolation Kit, for example, was realized that there was a way to tweak them,
enhanced for excellent recovery of human we didn’t hesitate to go the extra mile,” points
classical, non-classical, and intermediate out Dr. Claudia Loske, Product Manager,
Immunology.
monocyte subsets.
Besides ensuring outstanding separation Miltenyi Biotec is dedicated to continuously
performance, the protocols of various kits were advancing products that provide researchers
modified to save valuable time. Systematic with the best possible tools – stay tuned for the
optimization of the reagents allowed us to next updates.
minimize, for example, the time used for cell
If you have questions about the individual
kits and how they can enhance and accelerate
your research, contact our technical support
team. Get quick, helpful, local support from a
support team in your specific country. Contact
information is available at www.miltenyibiotec.
com/support. To get immediate support online,
use the Live Chat feature on the starting page
of our website.
Vol 15 • 1/2013
MACS & more 5
NEWS
As fast as it gets: cell isolation from
whole blood with MACSxpress® Technology
Isolating leukocytes from large whole blood samples with no time to lose – a challenge numerous labs face every day. Miltenyi Biotec
provides the solution: the new MACSxpress® Technology.
The MACSxpress CD4 T Cell Isolation Kit, human
enables isolation of untouched cells directly from
whole blood.
Fast, safe, and convenient
With the MACSxpress Cell Isolation Kits,
Miltenyi Biotec is breaking new ground. The
technology enables the fastest isolation of cells
from whole blood. “MACSxpress Technology
was specifically developed for cell isolation
from large whole blood volumes, in a short
time, and with great convenience,” explains
Volker Huppert, project leader at Miltenyi
Biotec’s R&D department. Using MACSxpress
Kits it takes only 20 minutes to prepare pure
leukocyte subsets from whole blood. This
means that the entire MACSxpress Cell
Isolation procedure is shorter than the sample
preparation step in traditional cell isolation
protocols. The short process opens up valuable
time for users to accomplish other tasks.
As the procedure does not involve any
centrifugation step or erythrocyte lysis, less
pipetting is required, and aerosol formation
is minimized. This high level of safety is
particularly relevant when processing untested
blood samples, which potentially contain
infectious particles. Minimal sample handling
also means maintenance of sample integrity
and a reduced risk for the user.
Large-scale isolation of leukocyte subsets
Studies often require numerous tests to be
conducted with the same sample. Such multiparameter assays require large numbers
of isolated cells, which are obtained using
MACSxpress Cell Isolation Kits. Using these
How it works
MACSxpress Cell Isolation is based on
a simple procedure: Erythrocytes are
aggregated and sedimented without
centrifugation. Non-target cells are
removed by immunomagnetic depletion
with MACSxpress Beads, yielding target
cells of high purity.
Simple yet powerful
MACSxpress Kits are the perfect solutions
for a wide range of applications, whether in
basic or translational immunology research.
The procedure involves only minimal
sample handling, which makes MACSxpress
Technology the ideal choice for omics studies,
biomarker research, or for drug discovery
research. Multicenter research studies can
also benefit from the new technology: “Users
don’t need any special expertise, equipment
requirements are minimal, and experiments
can be carried out in any lab in no time. The
simplicity of MACSxpress Technology is
compelling,” Volker Huppert points out.
MACSxpress Cell Isolation Kits are currently
available for B cells, naive B cells, NK cells, pan
T cells, CD4+ T cells, and CD8+ T cells.
Also read the report on page 10 of this
MACS&more issue: Mekes et al. used
MACSxpress Technology to isolate B cells for
sensitive gene expression analysis.
For general information on MACSxpress Kits
visit www.macsxpress.com
Cell isolation with MACSxpress Technology
Labeling
kits up to 30 mL of whole blood can be
processed in a single run. “Especially when
isolating cells with low frequencies, such as NK
cells, it is crucial to start with a large sample
volume in order to end up with a sufficient
number of cells for downstream analysis.
MACSxpress Kits meet these requirements
perfectly,” says Volker Huppert.
Separation
20 min
Density gradient centrifugation
Centrifugation without brake
35 min
0
10
20
30
Time (min)
MACSxpress Cell Isolation versus density gradient centrifugation. A leukocyte subset isolation with
MACSxpress Technology takes less time than a preparative density gradient centrifugation. With MACSxpress
Kits whole blood samples of up to 30 mL can be processed, whereas density gradient centrifugation has a
maximum capacity of 15 mL per sample.
6 MACS & more
Vol 15 • 1/2013
NEWS
Automated workflows in immunology research –
using simplicity to control complexity
Many research projects focusing on immune cell biology require a high sample throughput enabling complex, large-scale
experiments with the option to evaluate a high number of parameters simultaneously. Miltenyi Biotec and major liquid handling
system providers are working together to provide platforms for sophisticated automated workflows, including cell separation,
cell culture, and functional assays.
Facing the challenge
With the growing complexity of experiments
in today’s immunology research, the need
for high-throughput automated workflow
solutions increases. Workflow automation
ensures consistent and reliable results through
highly standardized, operator-independent
procedures. This is of particular relevance in
translational research, where multiple centers
work together towards a common goal. The
minimization of laborious manual handling
steps has many advantages and amenities.
Productivity increases, as experiments can
continue over night and on weekends, and,
instead of performing tedious pipetting steps,
researchers can focus on other tasks.
Automation is key
Miltenyi Biotec collaborates closely with major
providers of liquid handling systems (LHS),
including Tecan® and Hamilton Robotics,
towards the development of automated
workflows supporting most demanding
research applications. An example of a project the Mo-DC differentiation status utilizing
in the planning is a platform for automated the reagents of the Mo-DC Differentiation
monitoring of immune cell activity in the Inspector.
context of tumor formation. The platform “As complex the workflow may be, this platform
is based on the compelling synergy of LHS simplifies matters – the operator just starts
and Miltenyi Biotec instruments, including the process and walks away,” points out Dr.
the MultiMACS™ Cell24 Separator for cell Raif Yücel, Product Manager Automation
separation and the MACSQuant® Analyzer for at Miltenyi Biotec. “Automation not only
flow cytometric quality control and endpoint saves a lot of time, it also makes for excellent
analysis.
reproducibility.”
In the next step of the workflow, differentiated
Complex experiment workflow –
Mo-DCs are turned into tumor antigen–
walk-away processes
presenting cells by incubation with tumorThe automated workflow for monitoring specific peptides. Finally, the antigenimmune responses starts with the isolation presenting Mo-DCs are cocultured with the
of monocytes and T cells from PBMCs T cells, while the MACSQuant Analyzer, in
by the MultiMACS Cell24 Separator. The combination with MACS Cytokine Secretion
MACSQuant Analyzer then automatically Assays, determines the T cells’ cytokine
assesses purity and yield of the separated response. The magnitude of cytokine
cell populations. Isolated monocytes are production allows valuable conclusions on the
differentiated into monocyte-derived dendritic repertoire of tumor antigen-specific T cells in
cells (Mo-DCs) and T cells are cultured for the PBMCs.
later use. The MACSQuant Analyzer monitors “The huge versatility of the LHS and our
instruments, cell separation reagents,
antibodies, and cell culture products opens
up vast possibilities for basic and translational
research,” Dr. Yücel says. “Workflows can
be adapted to meet specific cell isolation
requirements, culture conditions, and assays.”
For more information visit
www.macslabautomation.com
The MultiMACS Cell24 Separator integrated into the LHS for automated high-throughput cell separation.
Vol 15 • 1/2013
MACS & more 7
NEWS
The CliniMACS® CD34 Reagent System –
a landmark in cellular therapy
Miltenyi Biotec strives for improvement of scientific understanding and medical progress by providing products and services that advance
biomedical research and cellular therapy. Working together with many internationally recognized professionals in centers throughout the
world, we design, support, and coordinate clinical studies in areas such as hematological malignancies and stem cell therapies.
AML is characterized by the rapid growth of
myeloid bone marrow cells. Transplantation of
allogeneic hematopoietic stem cells (HSCT) is
regarded as the single most effective treatment
for the prevention of reoccurrence of the
disease in patients in complete remission after
induction therapy.1,2
However, major complications are associated
with allogeneic stem cell transplantation, such
as acute and chronic graft-versus-host disease
(GvHD). Depending on the severity, GvHD
can be associated with increased morbidity and
mortality following transplantation.
Since both acute and chronic GvHD are
caused by the presence of donor T cells in
the graft, depletion of graft T cells (TCD)
before transplantation seemed to be a suitable
preventative strategy. However this strategy
was not universally accepted by clinicians
because of doubts about the efficacy of the
engineered graft.
The uncertainty associated with treatment with
HSCT was addressed by a recent study carried
out by researchers from the Blood and Marrow
Transplant Clinical Trials Network (Study
BMT CTN 0303). They reported that TCD and
HSCT, following intensive chemotherapy of
AML patients, can be performed reproducibly
in a multicenter setting using the CliniMACS
CD34 Reagent System.³,⁴ The data from the
BMT CTN 0303 study were compared to that of
a similar study in which patients had received
a hematopoietic stem cell graft that was not
T cell–depleted (BMT CTN 0101). The data
from this comparison demonstrated that TCD
with the CliniMACS CD34 Reagent System, as
the sole method for preventing GvHD, resulted
in a low incidence of acute and chronic GvHD
in patients. This method of TCD did not have
any negative impact on engraftment success,
patient survival, or relapse rate.⁵
8 MACS & more
Vol 15 • 1/2013
The effect on the occurrence of chronic GvHD
was especially notable: at the 2-year time point,
chronic GvHD occurred with a frequency of
only 19% in BMT CTN 0303 compared to 50%
in the study with the non-engineered graft, a
statistically significant difference.
Due to the highly efficient TCD there
was no need for any immunosuppressive
prophylaxis post transplantation, a feature
that may influence quality of life in a positive
way. Furthermore, the transplantation of a
T cell–depleted graft in the absence of an
immunosuppressive agent may provide an
ideal platform for further adoptive cell therapy
post transplantation.
3. Devine, S.M. et al. (2011) Biol. Blood Marrow
Transplant. 17: 1343–1351.
4. Keever-Taylor, C.A. et al. (2012) Biol. Blood
Marrow Transplant. 18: 690–697.
5. Pasquini, M.C. et al. (2012) J. Clin. Oncol. 30:
3194–3201.
The CliniMACS® CD34 Reagent System components,
Instrument, CD34 Reagent, Tubing Sets, and PBS/
EDTA Buffer, are manufactured and controlled under
an ISO 13485 certified quality system.
In Europe, the CliniMACS System components are
available as CE-marked medical devices.
In the USA, the CliniMACS System components,
including the CliniMACS Reagents, are available
for use only under an approved Investigational New
Drug (IND) application or Investigational Device
Exemption (IDE).
CliniMACS MicroBeads are for research use only and
not for use in humans.
References
1. Cornelissen, J.J. et al. (2007) Blood 109: 3658–
3666.
2. Koreth, J. et al. (2009) JAMA 301: 2349–2361.
Report
The CliniMACS® Prodigy
Next generation of integrated cell processing
Automated cell processing from cell sample to final product
• Cell washing
• Cell culture
• Density gradient separation
• Fully automated
• MACS® Cell Separation
• Closed system
At the time of publication, the components of the CliniMACS
Prodigy line are for research use only and not for human
therapeutic or diagnostic use. For an updated regulatory status
in your country, please ask your local representative.
clinimacs-prodigy.com
Vol 15 • 1/2013
MACS & more 9
Report
MACSxpress® Technology allows isolation
of B cells with high purity for sensitive
and concordant microarray-based
gene expression profiling
Angela Mekes, Stephanie Soltenborn, Ines Dischinger, Volker Huppert, and Bernhard Gerstmayer
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Introduction
Microarray-based gene expression profiling
is an excellent tool for the identification of
biomarkers in blood cells. However, whole
blood and PBMCs, which are frequently used
for biomarker research, are complex mixtures
of many different cell types and subsets.
Therefore, subtle changes in gene expression
within a particular subpopulation can escape
detection, if these complex mixtures are used
for analysis. There is also a large variability in
the proportions of blood cell subsets among
different donors, which can further mask
expression changes within a particular cell type.
The use of isolated blood cell subsets has
greatly enhanced biomarker research. Lyons
et al.¹ used purified leukocyte subsets isolated
by MACS® Technology for their biomarker
research in systemic lupus erythematosus.
The authors identified disease-associated
differentially expressed genes in purified CD4+
cells and monocytes that were not detectable
in PBMCs.
Similarly, Lee et al.² identified signatures in
magnetically purified CD8+ T cells in their
biomarker research on Crohn’s disease and
ulcerative colitis, which they otherwise would
not have detected in unseparated PBMCs.
10 MACS & more
Vol 15 • 1/2013
For omics research, in general, it is desirable
to have simple and short procedures that are
highly reproducible and require minimal
handling. The novel MACSxpress® Technology
for the fast isolation of cells directly from
whole blood meets all these requirements.
Using this technology, we isolated B cells from
different donors to high purity. The obtained
gene expression patterns were distinct from
the corresponding whole blood and PBMC
samples and showed enrichment of B cell–
specific gene transcripts as well as depletion of
non-B cell–related gene transcripts.
Materials and methods
B cell isolation
For the isolation of B cells, 30 mL of EDTAanticoagulated whole blood were incubated
with the MACSxpress B Cell Isolation Cocktail
for 5 min in a 50-mL tube. During the
incubation step, the tube was gently rotated
using the MACSmix™ Tube Rotator. The
B Cell Isolation Cocktail contains antibodyconjugated MACSxpress Beads magnetically
labeling the non-target cells. Following the
labeling step, the tube was placed in the
magnetic field of a MACSxpress Separator.
The labeled non-target cells adhered to the
tube wall and the aggregated erythrocytes
sedimented to the bottom, whereas the
supernatant contained pure unlabeled B cells,
which could immediately be used for further
experiments. Samples obtained from four
healthy donors were processed.
Flow cytometry
To assess the purity of B cells after isolation
with MACSxpress Technology, cells were
labeled with CD45-VioBlue®, CD19-APC, and
CD20-PE, before and after separation. Flow
cytometry was performed on the MACSQuant®
Analyzer. For comparison, PBMCs prepared by
density gradient centrifugation from the same
whole blood sample were analyzed.
Sample preparation for microarray analysis
RNA was extracted from whole blood
(stabilized with PAXgene® Blood RNA Tubes)
using the PAXgene Blood RNA Kit (Qiagen),
and from PBMCs and isolated B cells using the
NucleoSpin® RNA II system (Macherey-Nagel).
RNA quality, i.e., RNA integrity number (RIN),
was assessed using the Agilent 2100 Bioanalyzer
platform and the integrated software. For linear
T7-based amplification, 50 ng of total RNA
were used. Cy™3-labeled cRNA was prepared
Report
Whole blood
PBMCs
Isolated B cells
97.98%
CD19-APC
12.77%
CD19-APC
CD19-APC
7.43%
CD20-PE
CD20-PE
Donor
% B cells in
whole blood sample
% B cells
in PBMCs
% B cells after
MACSxpress Separation
A
4.27
10.85
93.2
C
5.06
10.01
97.06
D
7.43
12.77
97.98
E
5.76
10.95
90.82
means±sd
5.63±1.35
11.15±1.16
94.77±3.35
Figure 1 Flow cytometric analysis of whole blood, PBMCs, and isolated B cells. Cells were stained as indicated in the materials and methods section, and analyzed
on the MACSQuant Analyzer. Data were gated on leukocytes. Dot plots are shown for one representative donor. The table summarizes the results for samples from
four different donors.
by using the Agilent Low Input Quick Amp
Labeling Kit (Agilent Technologies) following
the manufacturer’s protocol.
Hybridization of Agilent
Whole Genome Oligo Microarrays
Hybridization was performed according to the
Agilent 60-mer oligo microarray processing
protocol using the Agilent Gene Expression
Hybridization Kit (Agilent Technologies).
Briefly, 1.65 μg of Cy3-labeled fragmented
cRNA in hybridization buffer were hybridized
overnight (17 hours, 65 °C) to Agilent Whole
Human Genome Oligo Microarrays 4×44K V1
using Agilent’s recommended hybridization
chamber and oven. Subsequently, the
microarrays were washed once with the Agilent
Gene Expression Wash Buffer 1 for 1 min at
room temperature followed by a second wash
with pre-heated Agilent Gene Expression Wash
Buffer 2 for 1 min at 37 °C. The last washing
step was performed with acetonitrile for 30 s at
room temperature.
Microarray analysis
Fluorescence signals of the hybridized Agilent
Microarrays were detected using Agilent’s
Microarray Scanner System (Agilent
Technologies). The Agilent Feature Extraction
Software was used to read out and process
the microarray image files. The software
determines feature intensities (including
background subtraction), rejects outliers, and
calculates statistical confidences.
Whole blood
PBMCs
Isolated B cells
RIN
7.93±0.19
8.63±0.25
8.18±0.51
RNA yield
(µg/mL whole blood)
2.6 ±1.96
0.77±0.34
0.064±0.012
Table 1 RIN values and yields for RNA extracted from different sample materials. RNA was extracted
as described in the materials and methods section. Values represent samples from four different donors
(means±sd).
Results and discussion
Isolation of B cells with
MACSxpress Technology
MACSxpress Technology allows the isolation
of B cells to excellent purities up to 98% (fig. 1).
The average purity was about 95% (n=4). The
recovery of B cells isolated from whole blood
ranged between 68% and 83%.
RNA quality
A RIN value of greater than 5 is considered
to indicate that RNA quality is sufficient for
gene expression profiling experiments³. In our
experiments, RNA prepared from whole blood,
PBMCs, and purified B cells consistently
showed RIN values of approximately 8. RNA
yields were sufficient for gene expression
profiling experiments (table 1).
Microarray analysis
Expression analysis of various cell markers in
whole blood vs. the purified B cell fraction
allowed us to further validate the purity of the
isolated B cells (fig. 2). As anticipated CD19+
cells were efficiently enriched, as indicated by
a 20-fold increase in light units (≈3,000 LU in
whole blood vs. ≈60,000 LU in purified B cells).
Vol 15 • 1/2013
MACS & more 11
Report
CD14
14,472
14,000
12,000
14,472
13,312
11,323
12,057
10,770
10,000
8,380
8,000
7,208
6,000
4,000
2,000
41
0
44
59
CD19
80,000
qn-normalized raw data
59
70,000
64,112
58,430
57,432
60,000
56,479
50,000
40,000
30,000
20,000
10,000
4,049
0
1,941
3,869
3,639
7,118
5,673
4,261
4,864
HBA1
500,000
450,000
400,000
444,292
425,889
444,292 444,292
350,000
300,000
250,000
200,000
150,000
100,000
50,000
0
157,853
0.0
135,816
28,235
16,610
10,203
1,733
5,120
4,054
MPO
1,600
1,400
1,335
1,000
0.7554416
800
600
585
467
400
318
186
200
127
1.0
224
107
20
23
48
3
3
2
25
2
PF4
15.0
0.51083195
1,200
0
1,000
900
800
700
600
500
400
300
200
100
0
939
392
291
121
107
76
79
23
TCL1A
6.102
A_Whole_blood
D_Whole_blood
C_Whole_blood
E_Whole_blood
A_PBMCs
D_PBMCs
C_PBMCs
E_PBMCs
A_Isolated_B_cells
D_Isolated_B_cells
C_Isolated_B_cells
E_Isolated_B_cells
16,000
Likewise, the lymphocyte-associated marker
TCL1A showed an increase from ≈10,000 LU
to 100,000 LU, whereas CD14 expression was
almost absent (≈10,000 LU vs. ≈50 LU). The
reticulocyte/erythrocyte–associated marker
HBA1 showed high values of ≈450,000 LU in
whole blood samples, which was in the range of
saturation, while the purified B cells showed a
reduced value of ≈5,000 LU. The values for the
neutrophil marker MPO were reduced from
≈250 LU to ≈30 LU, and the platelet marker
PF4 from ≈400 LU to ≈2 LU. These results
confirm that MACSxpress Technology allowed
the efficient removal of non-B cells from whole
blood.
We compared expression profiles of whole
blood samples, PBMCs, and purified B cells
in an unsupervised cluster analysis with 4,100
genes as input. Genes not associated with
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
161,605
143,146
119,744
83,508
9,865
11,234
A
C
17,269 12,168
D
E
Whole blood
9,567
11,798
15,584
10,849
A
C
D
E
PBMCs
A
C
D
E
Isolated B cells
Figure 2 Microarray-based analysis of cell type–specific gene expression. Gene expression analysis was
performed as indicated in the materials and methods section. Data indicate quantile-normalized raw data
(light units, LU) for whole blood samples, PBMCs, and isolated B cells, from four different donors (A,C,D,E)
each.
12 MACS & more
Vol 15 • 1/2013
SLA2
KLRD1
MAF
LEF1
SRGN
ENST00000390352
SIRPG
TRA@
ICOS
CD3E
UBASH3A
LAT
A_32_P180185
TXK
S1PR2
CD8B
FLJ22662
CD2
CD3G
C4orf18
CSTA
GIMAP7
PTGER2
GIMAP8
DUSP6
FCN2
FCN1
CD33
USP6NL
EBF1
MS4A1
FCRL2
IL7
COBLL1
BANK1
BLNK
CD19
L0C283663
AIM2
FCRLA
PNOC
CPNE5
WDR34
Figure 3 Gene expression profiling of cells
isolated by MACSxpress Technology. Gene
expression analysis was performed as indicated in the
materials and methods section. Data were extracted
from an unsupervised heatmap (input 4,100 genes)
and are shown for whole blood samples, PBMCs, and
isolated B cells, from four different donors each.
Report
B cells, such as CD3, CD8, and CD33, were not
represented in the purified B cell fraction. In
contrast, the B cell marker CD19 was highly
abundant in this fraction (fig. 3). Overall, the
heat map reveals considerable differences
between purified B cells and the unpurified
fractions (whole blood and PBMCs). A twoway analysis of variance showed that 2,111 of
4,100 genes were significantly differentially
expressed in purified B cells vs. unpurified cells.
This suggests that subtle yet significant gene
expression changes within the B cell fraction
might escape detection if unpurified cells are
used for analysis.
Conclusion
• Gene expression analysis of isolated cell
populations is more sensitive and conclusive
than analysis of whole blood or PBMCs.
• MACSxpress Technology is a fast method (as
little as 20 min) for the efficient isolation of
cells from large whole blood volumes (up to
30 mL) requiring minimal handling steps.
• Isolated B cells show high purities.
• The isolation procedure is gentle to cells. Cells
can be immediately used for downstream
applications.
• RNA extracted from isolated cells is of high
quality and suitable for gene expression
analysis.
• MACSxpress Technology is an ideal method
for the isolation of cells prior to gene
expression analysis.
References
1. Lyons, P.A. et al. (2010) Ann. Rheum. Dis. 69:
1208–1213.
2. Lee, J.C. et al. (2011) J. Clin. Invest. 121: 4171–
4179.
3. Fleige, S. and Pfaffl, M.W. (2006) Mol. Aspects
Med. 27: 126–139.
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human
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* Products and services are for research use only.
MACSxpress Cell Isolation Kits are also available for naive B cells,
NK cells, pan T cells, CD4+ T cells, and CD8+ T cells. For further
information visit www.macsxpress.com
For more information on Miltenyi Biotec's comprehensive genomic
and bioinformatic services visit www.macsgenomicservices.com
New
Effective activation of pDCs and B cells
High-quality oligonucleotides for TLR9 stimulation
DNA oligonucleotides (ODNs) containing the CpG
motif represent specific toll-like receptor 9 (TLR9)
ligands, enabling studies on the role of pDCs and
B cells in innate immunity and inflammation.
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activation of your specific cell type.
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Vol 15 • 1/2013
MACS & more 13
Report
Isolation of monocytes with high purity
directly from whole blood for transcriptome
analysis in translational research
Christelle Foucher1, Sébastien Vachenc1, Alexandre Meniccaci1, Bernhard Gerstmayer2, Gwenola Henrion1, Uwe Janssen2,
Darren Wilbraham3, Karine Le Malicot1, and Jean-Claude Ansquer1
Laboratoires Fournier SA, formely an Abbott Company, Daix, France
3
Quintiles Limited, London, UK
1
2
Introduction
Fenofibrate is a lipid-lowering drug used in
the treatment of dyslipidemia. Fenofibrate
effects have been attributed to the activation
of the nuclear transcription factor peroxisome
proliferator–activated receptor-α (PPARα).
PPARα plays a role in the regulation of tissue
factor expression in human monocytes and
might thus influence atherothrombosis¹,².
In this study, we assessed the effects of
fenofibrate on gene expression in peripheral
blood monocytes of healthy donors.
Microarray-based transcriptome analysis is
a powerful tool in translational research for
evaluating drug effects on particular blood
cell types. However, for the reliable detection
of subtle changes in gene expression in a
certain cell type, it is crucial to use isolated cell
populations of high purity for analysis.
In particular for translational research
projects involving multiple clinical centers
and operators, it is vital to use cell isolation
protocols that are short, simple, and highly
reproducible, and avoid steps that are prone to
variability, such as the preparation of peripheral
blood mononuclear cells (PBMCs). Positive
selection of CD14+ monocytes by MACS®
Technology is a well-established and reliable
procedure. Here we used the autoMACS® Pro
Separator and Whole Blood CD14 MicroBeads
to magnetically isolate monocytes directly from
whole blood and achieved high cell purities
and yields. This protocol avoids both PBMC
preparation and erythrocyte lysis, which are
14 MACS & more
Vol 15 • 1/2013
both laborious and can lead to variation in cell
separation results.
Microarray experiments and comparison
of gene expression at three different time
points of fenofibrate treatment allowed for
the identification of differentially expressed
sequences (DES) and modulated biological
functions. The experiment workflow is
summarized in figure 1.
Subjects, materials, and methods
Subjects and study design
Twenty six healthy males or post menopausal
(natural or chirurgical) females not receiving
hormone replacement therapy (HRT) or having
stopped HRT for at least 1 month, aged 40–65
years inclusive, were recruited in this openlabel, single-center research study to receive
a standard dose of fenofibrate as one 145 mg
tablet daily treatment (Lipanthyl®, Laboratoires
Whole blood samples from healthy donors
Isolation of monocytes with
autoMACS Pro Separator and
Whole Blood CD14 MicroBeads
Microarray analysis of purified
CD14+ monocytes
Figure 1 Workflow for the isolation of CD14+
monocytes directly from whole blood and
subsequent transcriptome analysis.
Fournier SA, Dijon, France). Subjects with a
body mass index (BMI) ≥30 kg/m2 or <18 kg/
m2 or with known hypersensitivity to fibrates,
or females with child bearing potential without
a reliable method of contraception, having
received an investigational drug in the last
90 days before date of inclusion were not
included in the study. All subjects had normal
folate levels (mean±sd: 15.2±11.1 ng/mL) and
vitamin B12 levels (404.1±165.2 pg/mL) at
inclusion in the study. The study included a
screening phase from a few days up to 3 weeks
(wks) and a treatment phase of 6 wks. Blood
was drawn and monocytes were separated at
baseline, after 7 to 10 days, and after 6 wks of
treatment.
Ethic approval was obtained from the Guy’s
Hospital Research Ethics Committee, London,
UK. Freely given informed consent was
obtained from each subject before enrollment.
Isolation of monocytes from whole blood
Freshly drawn whole blood (40 mL) was
anticoagulated using EDTA. Two aliquots
(15 mL each) were magnetically labeled with
Whole Blood CD14 MicroBeads. Labeled
CD14+ monocytes were automatically isolated
in two runs using the autoMACS Pro Separator
according to the manufacturer’s protocol.
Isolated monocytes from both runs were
combined and analyzed by flow cytometry
to determine viability, yield, and purity. For
subsequent RNA extraction, isolated cells were
centrifuged and flash-frozen.
Report
Statistical analysis
A global effect of the treatment on the 78
(26×3) intensity profiles was determined using
a 2-way ANOVA considering donors and
times of sample collection, adjusted by using
the Benjamini-Hochberg false-discovery rate
(FDR) method to adjust p values and control
for the first species error. Pairwise comparisons
of the DES between study visits were
performed using the Student-Newman-Keuls
(NK) test. A significant effect of the treatment
on the sequences was concluded for p ≤ 0.01.
For functional analysis, pathways and networks
were constructed based on the classification of
all the DES modulated in at least one pairwise
comparison (p ≤ 0.01 for NK test) via a
k-means clustering approach (user-defined
number of clusters = 11, cosine correlation and
centroid-based search). Functional networks
were constructed using the Ingenuity® Pathway
Analysis software (IPA version 8.6 build 93815).
Magnetic isolation of monocytes directly
from whole blood
CD14+ monocytes were enriched directly from
whole blood in an automated fashion using the
autoMACS Pro Separator and Whole Blood
CD14 MicroBeads. In the example shown in
figure 2 the frequency of monocytes in the
whole blood sample amounted to ≈6%. MACS
treatment. We identified 5,187 sequences that
were differentially modulated in at least one
of the pairwise comparisons. The distribution
was as follows: 3,924 DES between 0 and 1 wk,
1,973 DES between 0 and 6 wks, and 2,904 DES
between 1 and 6 wks of fenofibrate treatment
(fig. 3).
Quality and yield of RNA extracted from
isolated monocytes
The yield of monocytes magnetically isolated
from 30 mL of whole blood was high, which
allowed us to extract large amounts of RNA
for microarray analysis. The yield of RNA
extracted from isolated monocytes (n=78)
amounted to 2.15±0.73 µg (mean±sd). RIN
values reached 9.57±0.47 (mean±sd) indicating
consistently high RNA quality for sensitive,
reliable microarray experiments.
K-means clustering led to the identification of
11 clusters showing different trends over time
in gene expression after fenofibrate treatment.
One of the k-means clusters, which included
476 DES, showed a trend to down-regulation
after 1 wk and a trend to up-regulation
between 1 and 6 wks of treatment. Out of these
476 DES, 324 had mapped gene identities,
231 were eligible for networks, and 220 were
eligible for function pathway analysis around
immunological disease, cell-mediated immune
response, cellular assembly and organization,
cellular movement, antigen presentation,
cardiovascular disease, cellular growth and
proliferation, lipid metabolism, molecular
transport, inflammatory response etc. (fig. 4).
Microarray analysis of monocytes isolated
with MACS® Technology
We compared gene expression in monocytes
isolated from peripheral blood of healthy
donors at 0, 1, and 6 wks of fenofibrate
Before enrichment
After enrichment
6.15%
97.6%
CD14-PE
RNA extraction and microarray analysis
Total RNA was extracted from flash-frozen cell
samples using the NucleoSpin® RNA II system
(Macherey-Nagel). RNA was quantitated and
RIN values were assessed using the Agilent
2100 Bioanalyzer platform and the integrated
software. RNA was amplified and labeled with
Cy™3 using the Agilent Low Input Quick Amp
Labeling Kit (Agilent Technologies). RNA was
hybridized to Agilent Whole Human Genome
Oligo Microarrays (4×44K).
Technology allowed us to separate monocytes
to purities greater than 97% (fig. 2, dot plots).
The mean purity of monocytes in 78 samples
from 26 donors amounted to 94.1±6.17%. The
mean cell yield was 1.3×106±0.55×106. Purified
monocytes showed consistently high viabilities
of ≈96%. For details see the table in figure 2.
CD14-PE
Flow cytometry
Cells were labeled with CD14-PE and CD45FITC antibodies before and after separation,
and analyzed by flow cytometry. CD15
antibodies were used to evaluate the frequency
of granulocytes. All antibodies were obtained
from Miltenyi Biotec. Cell debris and dead
cells were excluded from the analysis based
on scatter signals and propidium iodide
fluorescence.
CD45-FITC
Parameter
CD45-FITC
Mean
sd
Frequency of monocytes in whole blood sample
(% among CD45+ cells)
8.1
2.25
Purity of monocytes after enrichment
(% among CD45+ cells)
94.1
Yield of enriched cells (number of cells per mL)
Viability of enriched cells (% live cells)
6.17
1.3×10
0.55×106
95.7
3.4
6
Figure 2 Isolation of CD14+ monocytes from whole blood using the autoMACS® Pro Separator and
Whole Blood CD14 MicroBeads. Cells were enriched as indicated in the subjects, materials, and methods
section. Before and after enrichment, cells were labeled with CD14-PE and CD45-FITC and analyzed by flow
cytometry.
Vol 15 • 1/2013
MACS & more 15
Report
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16 MACS & more
Vol 15 • 1/2013
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Report
Conclusion
• The autoMACS Pro Separator in combination
with Whole Blood CD14 MicroBeads allows
for rapid and robust magnetic isolation of
monocytes with high yields.
• Isolated CD14+ monocytes showed purities
of 94% on average.
• The use of isolated monocytes enables
sensitive and accurate microarray-based
transcriptome analysis of research samples.
• Significant short-term (1 wk) and middleterm (6 wks) effects of fenofibrate on gene
expression in monocytes were observed.
• Identification of a large number of DES
within a k-means cluster allowed for the
construction of functional networks with
eligible gene identities involved in particular
in metabolic and inflammatory pathways.
1070
240
973
203
1678
460
563
0 wks vs. 1 wk (3,924 DES)
0 wks vs. 6 wks (1,973 DES)
References
1. Marx, N. et al. (2001) Circulation 103: 213–219.
2. Neve, B.P. et al. (2001) Circulation 103: 207–212.
1 wk vs. 6 wks (2,904 DES)
Figure 3 Venn diagram for comparisons of DES at three time points of fenofibrate treatment.
The Student-Newman-Keuls algorithm was applied for all pairwise comparisons. For details see the subjects,
materials, and methods section.
0 vs. 1 wk
0 vs. 6 wks
1 vs. 6 wks
Figure 4 Functional networks constructed from a k-means cluster. Network-eligible genes from a cluster showing a trend to down-regulation after 1 wk and a
trend to up-regulation between 1 and 6 wks of fenofibrate treatment were used as “seeds” for in silico network generation. Down-regulation is indicated in green and
up-regulation in red. For details see the subjects, materials, and methods section.
MACS Product or Services*
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Whole Blood CD14 MicroBeads, human
CD14, CD15, and CD45 antibodies
Genomic services
130-090-879
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Vol 15 • 1/2013
MACS & more 17
REPORT
Report
An automated method for purification of
CD138+ cells from whole bone marrow samples
for multiple myeloma research
Hossain Mossafa and Sabine Defasque
Laboratoire Cerba, Département Génétique Cergy Pontoise, 95310 Saint Ouen L’Aumone, France
Introduction
and reliable automated isolation of CD138+ CD138 MicroBeads and the autoMACS Pro
Multiple myeloma (MM), the second most PCs from whole BM samples for MM research. Separator amounted to 95% in average (table
common hematological malignancy, is a
1). For the vast majority of MM samples (95%)
plasma cell (PC) disorder in the bone marrow Materials and methods
we obtained enough cells for the performance
(BM). MM is characterized by a large clinical Isolation of CD138+ cells
of the recommended panel of FISH analyses
heterogeneity despite the homogeneous For the isolation of CD138 + cells directly and genome-wide analysis.
morphological appearance of malignant PCs. from whole BM samples (1.5 to 3 mL), cells
Chromosomal aberrations are a hallmark were magnetically labeled with Whole Blood FISH analysis of CD138+ cells
of MM, and distinct genetic abnormalities CD138 MicroBeads (Miltenyi Biotec). Program CD138+ cell enrichment prior to FISH analysis
characterize the major subtypes of the disease. “posselwb” in combination with the “Clean” more than doubled the detection rate of
As an addition to interphase fluorescence in process was used for separation of CD138+ abnormalities (83% vs. 40% in unseparated
situ hybridization (FISH), the more global cells on the autoMACS Pro Separator (Miltenyi cells), and frequencies of abnormalities reached
assessment of the underlying cytogenetics by a Biotec) according to the manufacturer’s significant levels. Figure 1 shows examples
genome-wide analysis of malignant PCs, using protocol. Purities of isolated cell populations of FISH analyses of isolated CD138+ cells,
high-density, single-nucleotide polymorphism were determined by flow cytometry using comparing normal cells (left) with malignant
(SNP) arrays for molecular karyotyping, has CD138 and CD38 antibodies. The purity cells (right). The normal cell in figure 1A
significantly improved the detection and of DNA was analyzed using a NanoDrop™
Sample
Detectable PCs
CD138 + PCs
Instrument (Thermo Fisher Scientific).
identification of genetic lesions.
in bone marrow following
FISH and/or molecular karyotyping
samples (%)
isolation (%)
experiments with unseparated BM samples FISH analysis
1
2
86.3
have a 30 to 50% probability of showing false After separation CD138+ PCs were hybridized
2
1
88.9
results or failure due to technical reasons. This with the LSI D13S25 (13q14.3) Single Color
3
0
91.8
is especially true for samples from BM aspirates and/or the LSI IGH/FGFR3 Dual Color, Dual
4
5
93.0
that were affected by very low PC infiltration in Fusion Translocation Probe (t(4;14)(p16;q32)),
5
5
93.5
and/or the LSI P53 (17p13.1) Single Color
the BM.
Purified CD138+ cells are a prerequisite to Probe and analyzed by fluorescence microscopy.
6
16
93.7
increasing the sensitivity of FISH analysis or
7
11
95.5
SNP arrays. The quality of FISH and molecular Results
8
24
95.5
karyotyping results depends on the degree of Isolation of CD138+ cells directly from BM
9
25
97.3
samples
PC purity and DNA integrity after isolation.
10
30
97.5
Given the daily demands of laboratories there In a study of 100 MM samples from whole BM,
is a need for an automated platform for the we evaluated the efficiency and performance of Table 1 Frequency of CD138+ PCs prior to and
preparation of pure CD138+ cell populations. separation, cell purity, and the quality of DNA after enrichment from BM samples using Whole
Blood CD138 MicroBeads and the autoMACS
Here we used Whole Blood CD138 MicroBeads after purification of CD138+ cells. Purities of Pro Separator. PC purity was determined by flow
and the autoMACS® Pro Separator for the fast CD138+ PCs after isolation with Whole Blood cytometry using CD138 and CD38 antibodies.
18 MACS & more
Vol 15 • 1/2013
Report
shows two orange signals representing the two
alleles of the D13S319 locus. The malignant
cell shows only one orange signal, due to a
deletion affecting locus D13S319 or monosomy
of chromosome 13.
The normal cell in figure 1B shows two
orange (FGFR3) and two green (IgH) signals.
The malignant cell (fig. 1B, right) shows an
abnormal signal pattern with one orange
(FGFR), one green (IGH), and three fusion
signals, resulting from the chromosomal
translocation (t(4;14) FGFR3/IgH).
NanoDrop™ Analysis of DNA extracted from
isolated CD138+ PCs revealed high purity (data
not shown).
Conclusion
As a routine MM research laboratory, we
receive numerous BM samples every day.
Isolation of CD138+ cells from these samples
is necessary to increase the sensitivity of
downstream assays, such as FISH analysis or
molecular karyotyping by SNP arrays. Since
2007, we have performed more than 6,000
PC isolations. This large number necessitates
a reliable, rapid, and standardized method,
A
B
allowing us to isolate CD138+ cells from
multiple samples in a convenient way, while
maintaining sample integrity. Whole Blood
CD138 MicroBeads in combination with the
autoMACS Pro Separator meet all of our lab’s
requirements. The autoMACS Pro Separator
allows the standardization of cell separation
processes and ensures a rapid handling of MM
samples.
• Whole Blood CD138 MicroBeads enable fast
isolation of CD138+ cells directly from BM
samples, thus minimizing hands-on time and
maximizing the yield of target cells.
• No sample preparation is required, such as
density gradient centrifugation or red blood
cell lysis.
• Purified CD138+ cells can be immediately
subjected to FISH or molecular analyses.
• The detection rate of chromosomal
abnormalities per sample in MM and PC
dyscrasia significantly improves when
analysis is performed on purified populations
of CD138+ PCs.
• The platform allows the generation of
reproducible and consistent results – even in
multi-user settings.
CD138+ plasma cells
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Myeloma: t(4;14) FGFR3/ lgH
Figure 1 FISH analysis of CD138+ cells. PCs were
isolated from whole BM using Whole Blood CD138
MicroBeads. Isolated cells were subjected to FISH
analysis with (A) LSI D13S25 Single Color Probe,
or (B) LSI IGH/FGFR3 Dual Color, Dual Fusion
Translocation Probe. In both pictures a normal cell
is shown on the left and a malignant, mutated cell
on the right.
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Vol 15 • 1/2013
MACS & more 19
Report
Highly purified peripheral blood γ/δ T cells
isolated by MACS® Technology respond
to NOD2 ligand
Lothar Marischen, Hans-Heinrich Oberg, Christian Peters, Sandra Ussat, Hoa Ly, Dieter Kabelitz, and Daniela Wesch
Institute of Immunology, University of Kiel, Kiel, Germany
Introduction
(NOD) 2 was expressed in freshly isolated γ/δ Materials and methods
CD3 +γ/δ T cells display characteristics T cells⁸.
Cell isolation
of the adaptive and the innate immune Our studies revealed that highly purified γ/δ Human γ/δ T cells were depleted from
system¹. The dominant subset of γ/δ T cells express NOD2 mRNA and NOD2 PBMCs using the Anti-TCRγ/δ MicroBead
T cells expresses a Vγ9Vδ2 T cell receptor protein⁹. Furthermore, we investigated a Kit (Miltenyi Biotec). For depletion of human
(TCR), which recognizes phosphorylated possible function of NOD2 in freshly isolated CD14 + monocytes, CD14 MicroBeads
intermediates of the bacterial non-mevalonate γ/δ T cells by stimulating the cells with the (Miltenyi Biotec) were used. Cell separation
isoprenoid biosynthesis pathway. Synthetic minimally bioactive motif muramyl dipeptide was performed using LS Columns (Miltenyi
Biotec). After depletion, the proportion of
phosphoantigens such as bromohydrin (MDP) that mimics bacterial peptidoglycan.
residual γ/δ T cells or monocytes was <0.1%.
pyrophosphate (BrHPP) are capable of
inducing large-scale Vγ9Vδ2 T cell expansion,
which is independent of processing and
presentation of such molecules by classical
cross-linked TCR
MHC molecules¹,². After TCR stimulation, γ/δ
T cells rapidly release cytokines, such as IFN-γ
Cytokine
medium
medium
MDP-LD
MDP-DD
and MIP-1α (CCL3), thereby activating other
cells of the immune system³. Additionally, γ/δ
IL-1β
T cells possess features of innate immune cells,
such as antigen-presenting capacity and express
pattern recognition receptors (PRR), including
GRO
Toll-like receptors (TLR)⁴,⁵. TLR recognize
a broad variety of structurally conserved
molecules derived from microbes. TLR
control
(-)
(+)
ligands have been shown to costimulate TCRactivated γ/δ T cells by enhancing production
of cytokines and chemokines⁶,⁷. Besides TLR,
another class of PRR, the nucleotide-binding
leucine-rich repeat receptors (NLR), was Figure 1 Production of IL-1β and GRO in response to TCR cross-linking and MDP-LD. γ/δ T cells were
purified by positive selection with MACS Technology. 4×10⁶ cells per well (24-well plate) were cultured in
detected in γ/δ T cell lines. However, these the absence or presence of rabbit anti-mouse Ig (cross-linked TCR) and MDP-LD or MDP-DD as indicated.
studies did not address whether the NLR Culture supernatants were analyzed for cytokines after 24 h. Positive and negative controls were included in
nucleotide-binding oligomerization domain the array.
20 MACS & more
Vol 15 • 1/2013
Report
To enrich γ/δ T cells or monocytes from
PBMCs, the Anti-TCRγ/δ MicroBead Kit
or CD14 MicroBeads were used. PBMCs
were pre-treated with FcR Blocking Reagent
(Miltenyi Biotec) to avoid non-specific
binding of antibodies (Abs) to FcR-bearing
cells. The purity of the positively selected
A
cells ranged between 83 and 97% when
only one LS Column was used for positive
selection. This pre-enrichment of γ/δ T cells
by MACS® Technology was necessary to avoid
time-consuming flow sorting of PBMCs. To
definitely ensure the depletion of residual
monocytes in the enriched γ/δ T cell fraction
B
Before
depletion
After depletion
of monocytes
SSC
After depletion
of γ/δ T cells
SSC
Before
depletion
FSC
0%
78.41 %
no stain
CD3
2.14 %
FSC
0%
γ/δ
C
CD14
D
200
4000
IFN-γ (pg/mL)
MIP-1α (pg/mL)
5000
3000
2000
1000
0
incl. γ/δ
w/o γ/δ
100
50
0
w/o γ/δ &
CD14
medium
150
MDP-LD
incl. γ/δ
w/o γ/δ
w/o γ/δ &
CD14
MDP-DD
Figure 2 MIP-1α and IFN-γ production by PBMCs depleted or not of γ/δ T cells and monocytes. (A, B)
Gates were set on lymphocytes (A, upper dot plots) or monocytes (B, upper dot plots) to determine γ/δ T cells
or monocytes among PBMCs, respectively, before and after depletion of the appropriate cell population. The
frequencies of CD3+TCRγ/δ+ T cells (A, lower dot plots) or CD14+ monocytes (B, lower dot plots) among
PBMCs of one representative donor before and after depletion of γ/δ T cells or monocytes, respectively,
are shown. (C, D) Undepleted PBMCs (incl. γ/δ), PBMCs depleted of γ/δ T cells (w/o γ/δ), and PBMCs
depleted of both γ/δ T cells and monocytes (w/o γ/δ & CD14) were stimulated with BrHPP and rIL-2 in the
absence (medium) or presence of active MDP-LD or inactive MDP-DD as indicated. 1.5×10⁵ cells per well
(96-well plates) were used throughout the experiment. Concentrations of MIP-1α (C) and IFN-γ (D) in the
supernatants were determined by ELISA after 24 h. Results of fig. 2D were reproduced from Marischen et al.
(ref. 9) with the permission of John Wiley and Sons. Results of one representative experiment out of four are
shown in figure 2 C and D.
and vice versa, a further purification step was
done by sorting with a FACSAria® cell sorter
(BD® Biosciences) or by using another LS
Column. The purity of the cells was then >99%.
For further details, please refer to Marischen
et al.⁹.
Activation of cells
PBMCs and γ/δ T cell- or monocyte-depleted
PBMCs were stimulated with the γ/δ T cell–
specific antigen BrHPP (200 nM, Innate
Pharma®) in the presence of rIL-2 (Novartis).
Positively selected γ/δ T cells or monocytes
were cultured in uncoated wells or wells coated
with 1 µg/mL rabbit anti-mouse Ig, which binds
to anti-TCRγ/δ mAb present on the γ/δ T cells
or CD14 mAb present on monocytes after
labeling with MACS MicroBeads. Additionally,
all cell cultures were incubated with 10 µg/mL
MDP-LD isomer (tlrl-mdp, Invivogen®) or as
a control with inactive MDP-DD isomer (tlrlmdpc; Invivogen) for 24 h without rIL-2 in
serum-free X-VIVO™15 medium (Lonza®). For
detailed culture conditions see reference 9.
Cytokine analysis
Cytokines were analyzed in cell culture
supernatants by using the RayBio® Human
Cytokine Antibody Array VI & 6.1 Map and
VII & 7.1 Map (Hoelzel Diagnostic), which
allows simultaneous detection of 2×60
cytokines and chemokines. Signals were
detected by chemiluminescence, followed
by semiquantitative analysis with the AIDA
software (Raytest). To determine the intensity,
local background was subtracted from each
value and normalized against the positive
controls of each membrane. IFN-γ and MIP1α were detected by DuoSet® sandwich ELISA
(R&D Systems) according to the manufacturer’s
instructions.
Flow cytometry
To determine the proportion of γ/δ T cells
among PBMCs, cells were labeled with antiCD3 mAb and anti-TCRγ/δ mAb. Monocytes
among PBMCs were analyzed after staining
with anti-CD14 mAb. Antibodies were
obtained from BD Biosciences.
Vol 15 • 1/2013
MACS & more 21
Report
83.1 %
no stain
B
no stain
A
99.9 %
89.1 %
99.7 %
γ/δ
C
CD14
D
600
IFN-γ (pg/mL)
MIP-1α (pg/mL)
4500
3000
1500
0
γ/δ
400
200
0
monocytes
medium
MDP-LD
γ/δ
monocytes
MDP-DD
Figure 3 Effects of MDP-LD on highly purified monocytes and γ/δ T cells. (A, B) The purities of γ/δ
T cells (A) or CD14+ monocytes (B) were determined after MACS Separation with one LS Column (A and B,
left dot plots) and after subsequent flow sorting (A and B, right dot plots), shown for one representative donor.
(C, D) Highly purified γ/δ T cells (1.5×10⁵) were stimulated through the TCR via immobilized rabbit antimouse Ab in the presence or absence of MDP-LD or MDP-DD, whereas monocytes (1.5×10⁵) were cultured
in medium or activated by MDP-LD or MDP-DD as indicated. Concentrations of MIP-1α (C) and IFN-γ (D)
in the supernatants were analyzed by ELISA after 24 h. Results of figure 3D were reproduced from Marischen
et al. (ref. 9) with the permission of John Wiley and Sons.
MDP enhanced cytokine secretion in
purified γ/δ T cells
γ/δ T cells were isolated from PBMCs from
three healthy donors by positive selection
using an LS Column. The purity of the cells
was between 83 and 97%. Purified γ/δ T cells
were cultured in medium or stimulated by
TCR cross-linking (via rabbit anti-mouse Ig)
in the absence or presence of MDP-LD or
MDP-DD for 24 h. IL-1β and GRO production
were measured by a human cytokine Ab
array. Figure 1 shows that MDP-LD induced
secretion of IL-1β and GRO by the purified
TCR-stimulated γ/δ T cells, whereas MDP-DD
had no effect. However, IL-1β and GRO are
produced mainly by monocytes, and not by γ/δ
T cells. This suggests that residual monocytes
were present in the γ/δ T cell population,
22 MACS & more
Vol 15 • 1/2013
which could be responsible for production of
these cytokines. Similar to our results, Lancioni
and colleagues observed that CD4+ T cells with
a purity of 97% differ from highly purified
CD4+ T cells with regard to their responses to
LPS.¹⁰ Therefore, we examined in more detail
γ/δ T cell– or monocyte-depleted PBMCs and
highly purified, freshly isolated γ/δ T cells or
monocytes.
Abrogation of TCR/MDP-LP–induced IFN-γ
production after depletion of γ/δ T cells
from PBMCs
PBMCs were completely depleted of γ/δ T cells
(fig. 2A) or monocytes (fig. 2B) by MACS
Technology. Undepleted PBMCs or PBMCs
depleted of γ/δ T cells or PBMCs depleted of
both γ/δ T cells and monocytes were stimulated
with BrHPP in the absence or presence of
MDP-LD or MDP-DD for 24 h (fig. 2C, D). In
these experiments, we analyzed the cytokine
MIP-1α, which can be secreted by both γ/δ
T cells and monocytes, and IFN-γ, which is
exclusively produced by T cells. We observed
that MDP-LP, but not inactive MDP-DD,
enhanced MIP-1α production in undepleted
PBMCs, but also in PBMCs depleted of γ/δ
T cells. The depletion of monocytes almost
abolished the secretion of MIP-1α suggesting
that MDP-LP–reactive monocytes were the
major producers of MIP-1α. Moreover, MDPLD, but not MDP-DD, enhanced IFN-γ
secretion in BrHPP-stimulated PBMCs, which
was abrogated after γ/δ T cell depletion (fig.
2D). These experiments indicate a moderate
direct costimulatory effect of MDP-LD on
γ/δ T cells, which was investigated in further
experiments using highly purified γ/δ T cells
from 18 healthy donors (see ref. 9).
MDP-LD directly increases IFN-γ
production in highly purified γ/δ T cells
From the same PBMCs shown in figure 2 as
well as from 3 additional healthy donors (see
ref. 9), γ/δ T cells and CD14+ monocytes were
isolated by a combination of MACS Separation
and flow sorting or by two consecutive MACS
Separations. We obtained highly purified
cells from both separation procedures. The
data for cells purified by the combination of
MACS Separation and flow sorting from one
representative donor are shown in figure 3A
and B. Highly purified monocytes stimulated
with MDP-LD produced higher levels of MIP1α than the control or cells incubated with
inactive MDP-DD (fig. 3C). Freshly isolated,
highly purified γ/δ T cells from the same donor
secreted IFN-γ in response to TCR crosslinking. IFN-γ secretion was slightly enhanced
in the presence of MDP-LD, but not with MDPDD (fig. 3D). Purified monocytes did not
produce IFN-γ under these culture conditions
(fig. 3D). Similar results were obtained after
two consecutive MACS Separations as shown
in figure 6 of reference 9.
Conclusion
• MACS Separation followed by flow sorting as
well as two consecutive MACS Separations
permit the isolation of highly purified γ/δ
T cells or CD14+ monocytes from PBMCs.
• The highly purified cell populations allow
functional studies on innate receptors, such
Report
as NOD2, expressed by freshly isolated γ/δ
T cells.
• In order to obtain PBMCs devoid of γ/δ
T cells and CD14+ monocytes, these cells were
completely removed by MACS Separation.
• The biologically active MDP-LD isomer, but
not the inactive MDP-DD isomer, enhanced
IFN-γ production by TCR-stimulated,
freshly isolated, highly purified γ/δ T cells,
further underscoring a role of γ/δ T cells in
anti-bacterial immunity.
Acknowledgment
We thank John Wiley and Sons for the
kind permission to reproduce figures from
Marischen et al. (ref. 9).
References
1. Hayday, A.C. (2000) Annu. Rev. Immunol. 18:
975–1026.
2. Espinosa, E. et al. (2001) J. Biol. Chem. 276:
18337–18344.
3. Wesch, D. et al. (2005) Curr. Med. Chem. – AntiInflammatory & Anti-Allergy Agents 4: 153–160.
4. Brandes, M. et al. (2005) Science 309: 264–268.
5. Wesch, D. et al. (2006) J. Immunol. 176: 1348–
1354.
6. Pietschmann, K. et al. (2009) Scand. J. Immunol.
70: 245–255.
7. Wesch, D. et al. (2011) Cell. Mol. Life Sci. 68:
2357–2370.
8. Hedges, J.F. et al. (2005) J. Immunol. 174: 6045–
6053.
9. Marischen, L. et al. (2011) Scand. J. Immunol. 74:
126–134.
10.Lancioni, C.L. et al. (2009) J. Immunol. Methods
344: 15–25.
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Vol 15 • 1/2013
MACS & more 23
Report
Efficient and rapid in vitro generation of fully
functional multi-virus-specific CD4+ and CD8+
T cells
Anna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer, Jürgen Schmitz, Mario Assenmacher, and Anne Richter
Introduction
Antigen-specific T cells play a critical role in
the regulation of immune responses and the
elimination of virus-infected or malignant cells
in the human body. T cells are essential, e.g.,
for the control of the outgrowth of EpsteinBarr virus (EBV)-infected B cells. CD4+ and
CD8+ T cells specific for the EBV antigens
BZLF1¹-⁴ and EBNA-1⁵-¹¹ have been found in
EBV-infected individuals. Virus-specific T cells
also hold great potential for clinical use. The
adoptive transfer of clinical-grade CD4+ and
CD8+ T cells specific for adenovirus (AdV)
hexon, cytomegalovirus (CMV) pp65, and EBV
antigens is a valuable tool for the treatment
of AdV¹², CMV¹³-¹⁶, and EBV¹⁷,¹⁸ infections
after hematopoietic stem cell transplantation.
To further enhance research on virus-specific
T cells, we have established a protocol for the
efficient and rapid generation of EBV-, AdV-,
and CMV-specific (tri-virus-specific) T cells.
The virus-specific CD4+ and CD8+ T cells were
stimulated in vitro using peptide pools, and
subsequently magnetically enriched according
to their IFN-γ secretion. The tri-virus-specific
T cell population could be easily expanded
without major alterations in the proportions of
the respective specificities.
Generation of multi-virus-specific T cells
For the generation of multi-virus-specific
T cells we stimulated 10⁹ PBMCs from
leukapheresis products of several healthy
donors with a combination of four PepTivator®
Peptide Pools (Miltenyi Biotec) covering CMV
pp65 or IE-1, AdV hexon, or EBV EBNA-1
or BZLF-1 for four hours. The simultaneous
addition of four peptide pools to a single
Generation of multi-virus-specific T cells
PBMCs
Mixed
antigen loading
Separate
antigen loading
PBMCs
PBMCs
Stimulation with
a mix of antigens
1+2+3+4
1/4
Loading
antigen 1
1/4
Loading
antigen 2
1/4
Loading
antigen 3
1/4
Loading
antigen 4
Stimulation
Enrichment of IFN‑γ–
secreting T cells
Enrichment of IFN-γ–secreting T cells
T cell expansion
T cell expansion
Analysis for specificities
Intracellular cytokine staining after in vitro restimulation
with a mixture and individual peptide pools
Figure 1 Experimental procedure for the generation of multi-virus-specific T cells. For details see the
materials and methods section.
24 MACS & more
Vol 15 • 1/2013
Report
1.4×10⁶
0.42 %*
98.6 %*
Before
enrichment
After
enrichment
0.46 %*
CD4
98.4 %*
1.45 %*
1.80 %*
99.5 %*
CD8
CD8
99.8 %*
1.0×10⁶
1×10⁷
LPLP
3/13/1
mix
mix
LPLP
3/13/1
separate
separate
Number of enriched IFN-γ+ T cells
per 10⁹ lymphocytes
After
enrichment
Separate antigen loading
Number of IFN-γ+ T cells out of 10⁹ lymphocytes
Before
enrichment
B
1.2×10⁶
Mixed antigen loading
CD4
A
LPLP
19/2
mix
19/2
8.0×10⁵
mix
LPLP
19/2
separate
19/2
separate
LPLP
6/26/2
mix
6.0×10⁵
mix
LPLP
6/26/2
separate
separate
LPLP
11/2
mix mix
11/2
LPLP
11/2
separate
11/2
separate
1×10⁶
4.0×10⁵
2.0×10⁵
1×10⁵
1.0×10³
CD8+ T cells
CD4+ T cells
1×10⁴
CD4+
T cells
secreted IFN-γ
secreted IFN-γ
* among CD4+/CD8+ T cells
CD8+
T cells
Figure 2 Enrichment of multi-virus-specific T cells after stimulation with individual peptide pools or a combination thereof. PBMCs from four different
donors were stimulated in two different ways as described in the materials and methods section: i) Cells were stimulated with a combination of pp65, IE-1, hexon,
and BZLF1 peptide pools in a single batch (mixed antigen loading), or ii) cells were loaded with the individual peptide pools in four aliquots, and aliquots were
subsequently combined for further stimulation (separate antigen loading). Subsequently, virus-specific T cells were enriched according to IFN-γ secretion. Before and
after enrichment IFN-γ–secreting CD4+ and CD8+ T cells were analyzed by flow cytometry. (A) Results from one representative donor are shown. Numbers indicate
frequencies among CD4+ or CD8+ cells. (B) Numbers of IFN-γ–positive CD4+ and CD8+ T cells enriched from PBMCs of four different donors.
A
select antigen-activated IFN-γ–secreting CD4+
and CD8+ T cells. Purities of enriched cells
were determined by flow cytometry using
the MACSQuant® Analyzer (Miltenyi Biotec).
Enriched multi-virus-specific CD4+ and CD8+
T cells were expanded in the presence of IL‑2
and feeder cells for 9–13 days. A flowchart
illustrating the experimental procedure is
shown in figure 1.
Mixed antigen loading
Unstimulated
sample
Separate antigen loading
Stimulated
sample
31.39%*
0.04%*
B
100
Stimulated
sample
LP 3/1 mix
LP 3/1 separate
LP 19/2 mix
LP 19/2 separate
LP 6/2 mix
LP 6/2 separate
LP 11/2 mix
LP 11/2 separate
90
51.40%*
CD4
CD4
0.11%*
Unstimulated
sample
Analysis of PBMCs and multi-virus-specific
T cells for antigen specificity
For the analysis of antigen specificity and
functionality of the enriched, expanded multivirus-specific T cells, we examined the IFN-γ
response after short-term in vitro restimulation
with individual peptide pools or a mixture
thereof. For comparison, we also restimulated
PBMCs. The IFN-γ response was determined
by intracellular cytokine staining using an
80
% IFN-γ+ T cells
sample might decrease the activation efficacy
for each peptide pool due to competition of
peptides for MHC binding. As a control, we
divided the PBMC samples into four aliquots,
incubated each aliquot with a single peptide
pool for two hours, and recombined the four
aliquots for T cell stimulation for another
four hours. Afterwards, we used the Large
Scale IFN-γ Secretion Assay – Enrichment Kit,
human, from Miltenyi Biotec to magnetically
70
60
+_ T cells
50
40
% IFN-
33.99%*
0.08%*
CD8
CD8
0.03%*
61.74%*
30
20
10
0
intracellular IFN-γ
CD4+
T cells
CD8+
T cells
* among CD4+/CD8+ T cells
Figure 3 Restimulation of enriched and expanded multi-virus-specific T cells. Enriched IFN-γ–secreting cells were expanded for 9 to 13 days. Subsequently, cells
were restimulated with a mixture of peptide pools or were left unstimulated. Cells were analyzed for IFN-γ production by intracellular staining and flow cytometry. (A)
Results from one representative donor are shown. Numbers indicate frequencies among CD4+ and CD8+ T cells, respectively. (B) Frequencies of IFN-γ–positive CD4+
and CD8+ T cells. Results from four different donors are shown.
Vol 15 • 1/2013
MACS & more 25
Report
donor 3/1
donor 6/2
donor 19/2
donor 11/2
100
90
80
80
BZLF1
60
EBNA-1
AdV hexon
50
IE-1
pp6540
30
20
10
0
PBMC
mix separate
PBMC
mix separate
PBMC
mix separate
PBMC
IE-1
pp65
BZLF1
EBNA-1
AdV hexon
IE-1
pp65
mix separate
20
10
PBMC
mix separate
PBMC
mix separate
PBMC
mix separate
PBMC
mix separate
mix
0
separate
mix
mix separate
pp65
30
separate
PBMC
PBMC
mix
mix separate
separate
PBMC
PBMC
mix separate
mix
PBMC
separate
0
IE-1
40
PBMC
10
50
mix
20
BZLF1
AdV hexonEBNA-1
AdV hexon
separate
30
60
PBMC
40
BZLF1
EBNA-1
mix
50
PBMC
60
donor 11/2
donor
11/2
donor 6/2
donor
6/2
80
separate
+
80
90
donor 19/2
donor
19/2
PBMC
80
100
separate
90
donor 3/1
donor
3/1
80
Relative frequency of specific CD4+ T cells (%)
donor 11/2
donor
11/2
mix
100
donor 19/2
donor
19/2
+
Relative
frequency
specific
T cells (%)
Relative
frequencyof
of specific
CD8CD8
T cells (%)
donor 3/1
donor
3/1
PBMC
+
+
cells (%)
RelativeRelative
frequency
frequencyof
ofspecific
specific CD4CD4
T cellsT(%)
A
B
Number of
IFNγ+CD4+ T cells
1×10⁸
CMV pp65
CMV
pp65
CMV IE-1
CMV
IE-1
1×10⁸
1×10⁷
1×10⁶
1×10⁷
1×10⁶
1×10⁵
1×10⁶
1×10⁵
1×10⁴
1×10⁵
1×10³
1×10⁴
PBMC
Number of
IFNγ+CD8+ T cells
1×10⁷
1×10⁴
PBMC
after
expansion
AdV hexon
AdV
hexon
after
expansion
1×10⁶
EBV EBNA-1
EBV
EBNA-1
1×10⁵
1×10⁵
1×10⁴
1×10⁴
after
expansion
PBMC
after
expansion
1×10⁸
1×10⁷
1×10⁷
1×10⁷
1×10⁷
1×10⁷
1×10⁶
1×10⁶
1×10⁶
1×10⁶
1×10⁶
1×10⁵
1×10⁵
1×10⁵
1×10⁵
1×10⁵
1×10⁴
1×10⁴
1×10⁴
1×10⁴
1×10⁴
PBMC
after
expansion
1×10³
PBMC
after
expansion
1×10³
PBMC
after
expansion
1×10³
EBV BZLF1
EBV
BZLF1
LP 11/2 mix
LP 11/2 separate
LP 6/2 mix
LP 6/2 separate
LP 19/2 mix
LP 19/2 separate
1×10³
1×10³
PBMC
1×10⁶
PBMC
after
expansion
1×10³
PBMC
after
expansion
PBMC
after
expansion
LP 3/1mix
LP 3/1 separate
Figure 4 Analysis of individual virus-specific T cell populations. Multi-virus-specific T cells were enriched as indicated in figure 2 and expanded. PBMCs and
multi-virus-specific T cell lines were restimulated with individual peptide pools or a mixture thereof. The relative frequencies and absolute cell numbers of T cells with
a single antigen specificity were calculated based on total cell numbers and the frequencies of IFN-γ–positive T cells among PBMCs and multi-virus-specific T cells
upon restimulation with individual peptide pools. Data from different donors are shown as indicated.
Anti-IFN-γ-PE antibody (Miltenyi Biotec).
CD4+ and CD8+ cells were then analyzed
by flow cytometry using the MACSQuant
Analyzer. Based on total cell numbers and the
frequencies of IFN-γ–positive T cells among
PBMCs and multi-virus-specific T cells upon
restimulation with individual peptide pools, we
calculated the relative frequencies and absolute
cell numbers of T cells with a single antigen
specificity.
26 MACS & more
Vol 15 • 1/2013
Magnetic enrichment of multi-virusspecific CD4+ and CD8+ T cells
Using the Large Scale IFN-γ Secretion Assay –
Enrichment Kit, we were able to consistently
enrich virus-specific CD4+ and CD8+ T cells
to purities higher than 90%. Flow cytometric
analyses of IFN-γ–secreting CD4+ and CD8+
T cells in PBMCs before and after magnetic
enrichment are shown in figure 2A for one
representative donor. Prior to enrichment, cells
were stimulated in two different ways: Cells
were either stimulated with a combination of
pp65, IE-1, hexon, and BZLF1 peptide pools
in a single batch, or cells were loaded with
the individual peptide pools in four aliquots,
and aliquots were subsequently combined
for further stimulation. Both stimulation
procedures resulted in comparable frequencies
of IFN-γ–secreting T cells before enrichment
Report
(fig. 2A). Likewise, purities of enriched cells
(fig. 2A and data not shown) and numbers
of enriched cells (fig. 2B) were similar in the
respective samples from all tested donors,
regardless of whether the samples underwent
mixed or separate antigen loading.
Specificity and functionality of the enriched
and expanded multi-virus-specific T cells
Within 9 to 13 days cell populations that
were generated by either mixed or separate
antigen loading expanded between 25- and
496-fold (data not shown). Both cell lines
were restimulated with a mixture of peptide
pools and analyzed for intracellular IFN-γ
production. The percentage of IFN-γ–reexpressing CD4+ T cells derived from mixed
antigen loading and separate antigen loading
amounted to 23.8–90.5% and 32.5–84.8%,
respectively. Similarly, the percentage of CD8+
T cells amounted to 31.8–88.0% and 34.0–
79.2%, respectively (fig. 3A,B). These results
confirm the high specificity and functionality
of the T cell lines. T cell expansion rates and
frequencies of T cells re-expressing IFN-γ were
similar, regardless of whether the cell lines
were originally generated by mixed or separate
antigen loading (fig. 3B).
Individual virus-specific T cell populations
show similar enrichment and expansion
rates
The strategy for the enrichment of virusspecific T cells from PBMCs is based on IFN-γ
expression induced by stimulation with the
peptide pools. Therefore, differences between
stimulation efficacies of individual peptide
pools would result in differences in IFN-γ
responses of the respective virus-specific
T cells, and ultimately in a biased proportion
of specificities within the enriched multivirus-specific T cell population. To ensure that
the proportions of the different specificities
are similar before and after enrichment, we
compared the relative frequencies (fig. 4A) and
absolute numbers (fig. 4B) of T cells specific
for each single antigen in PBMCs and in the
T cell lines. The relative frequencies of T cells
specific for the individual CMV, EBV, and AdV
antigens were about the same in PBMCs and
in both T cell lines. This demonstrates that all
individual virus-specific T cell populations
within PBMCs were effectively stimulated and
enriched. Moreover, regardless of whether the
original PBMCs were loaded with a mixture
of peptide pools or separately with single
peptide pools, the obtained T cell lines were
comparable with respect to the expansion rate
of the individual specificities.
Conclusion
• A combination of several PepTivator Peptide
Pools enables the simultaneous, effective
activation of CMV-, EBV-, and AdV-specific
(tri-virus-specific) CD4+ and CD8+ T cells.
• Activated tri-virus-specific T cells can be
co-enriched using the Large Scale IFN-γ
Secretion Assay – Enrichment Kit.
• Enriched tri-virus-specific T cells can be
expanded without significantly altering the
proportion of the individual specificities.
• Enriched tri-virus-specific T cells are fully
functional and re-express IFN-γ upon
restimulation.
References
1. Bogedain, C. et al. (1995) J. Virol. 69: 4872–4879.
2. Saulquin, X. et al. (2000) Eur. J. Immunol. 30:
2531–2539.
3. Precopio, M.L. et al. (2003) J. Immunol. 170:
2590–2598.
4. Tynan, F.E. et al. (2005) J. Exp. Med. 202: 1249–
1260.
5. Blake, N. et al. (2000) J. Immunol. 165: 7078–7087.
6. Khanna, R. et al. (1995) Virology 214: 633–637.
7. Paludan, C. et al. (2002) J. Immunol. 169: 1593–
1603.
8. Leen, A. et al. (2001) J. Virol. 75: 8649–8659.
9. Voo, K.S. et al. (2004) J. Exp. Med. 199: 459–470.
10.Voo, K.S. et al. (2002) Cancer Res. 62: 7195–7199.
11.Long, H.M. et al. (2005) J. Virol. 79: 4896–4907.
12.Feuchtinger, T. et al. (2006) Br. J. Haematol. 134:
64–76.
13.Feuchtinger, T. et al. (2010) Blood 116: 4360–4367.
14.Einsele, H. et al. (2002) Blood 99: 3916–3922.
15.Mackinnon, S. et al. (2008) Blood Cells Mol. Dis.
40: 63–67.
16.Peggs, K.S. et al. (2011) Clin. Infect. Dis. 52: 49–
57.
17. Moosmann, A. et al. (2010) Blood 115: 2960–2970.
18. Icheva, V. et al. (2013) J. Clin. Oncol. 31: 39–48.
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PepTivator AdV5 Hexon, human
PepTivator EBV EBNA-1, human
PepTivator EBV BZLF1, human
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IFN-γ Secretion Assay – Cell Enrichment and Detection Kit (PE),
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Human IL-2 and other cytokines
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Vol 15 • 1/2013
MACS & more 27
Report
Mouse NK cells isolated to high purity by
MACS® Technology are fully functional
Kathrin Meinhardt1, Irena Kroeger1, Sabine Mueller2, and Evelyn Ullrich1,3,4
1
2
3
4
Department of Internal Medicine 5 – Hematology/Oncology, University of Erlangen, Germany
Laboratory for Cellular Immunology, Pediatric Hematology & Oncology, J. W. Goethe University, Frankfurt am Main, Germany
Center for Cell and Gene Therapy, Frankfurt am Main, Germany
Introduction
% of NK cells
MicroBeads, mouse (Miltenyi Biotec) in
NK cells play a crucial role in both innate combination with the autoMACS® Pro
and adaptive immunity. Due to their capacity Separator (Miltenyi Biotec) and the program
to lyse tumor cells without pre-activation, “PosselD” according to the standard protocol.
they hold great potential for use in cellular Untouched NK cells were isolated using
therapies¹. To investigate the function of NK the NK Cell Isolation Kit, mouse (Miltenyi
cells in detail, it is crucial to work with cell Biotec) and the autoMACS Pro Separator with
populations of high purity. In this study, we program “DepleteS”. We further improved the
compared two different strategies for NK cell depletion cocktail included in this kit to allow
isolation based on MACS® Technology: i)
positive selection of NK cells according to the
expression of CD49b (DX5) and ii) depletion
of non-NK cells. Both approaches yielded NK
Spleen
cells with high purities from BALB/c mouse
10
spleen. We further optimized the procedure
***
8
for the depletion of non-NK cells, allowing for
the isolation of highly pure NK cells also from
6
C57BL/6 mouse spleen. Isolated NK cells were
fully functional in terms of IFN-γ secretion
4
and tumor lysis capacity.
Sample preparation
Spleens from female, 7–9-week-old C57BL/6
and BALB/c mice were dissociated into singlecell suspensions as described².
Isolation of NK cells
CD49b (DX5)+ NK cells were isolated by
positive selection using CD49b (DX5)
28 MACS & more
Vol 15 • 1/2013
for a more effective depletion of residual T
cells, B cells, and macrophages. This optimized
cocktail is now part of the protocol of the new
NK Cell Isolation Kit II from Miltenyi Biotec.
For NK cell isolation from C57BL/6 spleens,
we recommend incubation periods of 10–15
minutes with antibodies and MicroBeads.
Purified cell populations were subsequently
analyzed by flow cytometry.
15
Blood
*
10
2
2
0
BALB/c
C57BL/6
0
BALB/c
C57BL/6
Figure 1 Frequencies of NK cells in spleen and peripheral blood from two different mouse strains.
NK cells from spleens (n=7) and blood (n=4) from BALB/c and C57BL/6 mice were analyzed by flow cytometry
with gating on viable CD49b+NKp46+ cells. ***p=0.0006, *p=0.0286. Data were adapted from reference 2.
Report
NK cell frequencies in spleen and peripheral
blood from different mouse strains
To gain insight into the overall NK cell
content of mouse spleen and peripheral
blood from the mouse strains C57BL/6 and
BALB/c, we analyzed the frequency of CD3–
CD49b+NKp46+ NK cells by flow cytometry.
Figure 1 indicates that the percentage of NK
cells in both spleen and blood from BALB/c
mice was approximately twice as high as from
C57BL/6 mice. There were no significant
differences in the proportions of CD27+CD11b–,
CD27+CD11b+, and CD27–CD11b+ NK cell
subsets.²
Magnetic isolation of splenic NK cells
For detailed characterization of NK cells, in
particular functional analysis, it is crucial to use
cell separation methods that allow for high cell
purities. Here we compared different strategies
based on MACS Technology for the isolation of
NK cells from BALB/c and C57BL/6 mice. The
first strategy involved positive selection of NK
cells using CD49b (DX5) MicroBeads, based
on the expression of the CD49b antigen. The
second approach utilized depletion of non-NK
cells using the NK Cell Isolation Kit, mouse,
resulting in the isolation of untouched NK cells.
Figure 2 shows that both approaches allowed
CD49b
NK Cell
Isolation Kit
NK Cell
Isolation Kit II
81% +/– 2
90% +/– 3
94.9% +/– 1.3
65% +/– 5
77% +/– 10
89.6% +/– 3.1
BALB/c
C57BL/6
NKp46
Figure 2 Isolation of splenic NK cells from BALB/c and C57BL/6 mice using different strategies based
on MACS Technology. Cells were isolated as described in the materials and methods section. Subsequently,
CD49b (DX5)+NKp46+ cells were analyzed by flow cytometry. Numbers indicate mean purities and standard
deviations from at least five independent experiments. Data were adapted from reference 2.
for efficient isolation of NK cells from BALB/c
mice with purities of up to 90%, whereas cells
from C57BL/6 mice reached lower purities of
up to 77%. This led us to modify the depletion
cocktail to achieve a more effective removal
of residual T cells, B cells, and macrophages.
Strikingly, the new depletion cocktail, i.e., NK
Cell Isolation Kit II, resulted in high-purity NK
BALB/c
C57BL/6
2000
500
400
1500
IFN-γ (pg/mL)
Tumor lysis assay
The capacity of isolated NK cells to lyse tumor
cells was analyzed by using the crystal violet
assay as described previously¹. Briefly, 5,000
tumor cells were plated in 96-well plates
and cocultured with isolated NK cells at an
effector:target ratio of 10:1. To avoid MHC
mismatch effects, we used B16F10 melanoma
cells together with NK cells from C57BL/6
mice, and CT26 colon carcinoma cells with NK
cells from BALB/c mice. The tumor cells were
originally derived from the same background
as the corresponding NK cells.
CD49b (DX5)
MicroBeads
IFN-γ (pg/mL)
Analysis of cytokine production by
isolated NK cells
To assess the function of isolated NK cells, we
determined their capacity to secrete IFN-γ. To
this end, the cells were stimulated overnight
with IL-2 (50,000 U/mL). The next day, cell
culture supernatants were analyzed using the
BD® OptEIA™ Mouse IFN-γ ELISA Set (BD
Biosciences).
1000
500
300
200
100
0
0
CD49b (DX5) MicroBeads
NK Cell Isolation Kit
NK Cell Isolation Kit II
Figure 3 IFN-γ production by isolated NK cells. Cells were stimulated with IL-2 (50,000 Units/mL) over
night. Cell culture supernatants were analyzed by an IFN-γ–specific ELISA. One representative experiment
of at least three independent experiments is shown. Experiments were performed in triplicates. Data were
adapted from reference 2.
Vol 15 • 1/2013
MACS & more 29
Report
BALB/c
80
100
% of specific tumor lysis
% of specific tumor lysis
100
C57BL/6
CT26
60
40
20
0
B16F10
80
60
40
20
0
CD49b (DX5) MicroBeads
NK Cell Isolation Kit
NK Cell Isolation Kit II
Figure 4 Tumor lysis assay. For the crystal violet assay, 5,000 tumor cells were plated in 96-well plates and
cocultured with isolated NK cells at an effector: target ratio of 10:1. One representative experiment of at least
three independent experiments is shown. Experiments were performed in triplicates. Data were adapted from
reference 2.
cells from both C57BL/6 (approx. 90%) and Conclusion
BALB/c spleens (approx. 95%). Importantly, • Both CD49b (DX5) MicroBeads and the NK
Cell Isolation Kit allow for the isolation of NK
the subset distribution of CD27+CD11b–,
CD27 +CD11b +, and CD27 –CD11b + cells
cells from BALB/c mouse spleens with high
remained unaltered, regardless of whether
purity.
the cells were isolated by positive selection or • The new NK Cell Isolation Kit II yields
depletion (not shown).
highly pure NK cells from BALB/c mice
and represents the optimal solution for the
Functional analysis of isolated NK cells
isolation of C57BL/6 NK cells.
Next we tested whether the isolated cells • Isolated NK cells are fully functional
retained their full functionality, i.e., the
regardless of whether they were isolated by
capacity to produce cytokines in response to
positive selection or depletion.
stimulation with IL-2. Our results indicate
that both BALB/c and C57BL/6 NK cells from References
1. Terme, M. et al. (2008) Nat. Immunol. 9: 486–494.
all three isolation procedures secreted IFN-γ
2. Meinhardt, K. et al. (2012) J. Immunol. Methods
(fig. 3). NK cells prepared with the NK Cell
378: 1–10.
Isolation Kit II showed slightly reduced IFN-γ
production compared to the other approaches.
This may be due to the higher NK cell purity in
these preparations and thus a lower proportion
of contaminating cells that might influence the
IFN-γ production.
Moreover, we analyzed the isolated NK cells for
their capacity to lyse tumor cells. Our results
MACS Product*
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from the crystal-violet assay show that BALB/c
and C57BL/6 NK cells effectively lysed CT26
autoMACS Pro Separator –
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Starter Kit
colon carcinoma and B16F10 melanoma cells,
respectively (fig. 4).
CD49b (DX5) MicroBeads,
130-052-501
mouse
NK Cell Isolation Kit II, mouse
130-096-892
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in vivo applications. The products are
manufactured and tested under a certified
ISO 9001 quality system and in compliance
with relevant GMP guidelines. They are
designed following the recommendations
of USP <1043> on ancillary materials.
30 MACS & more
Vol 15 • 1/2013
Report
Measuring prolyl aminopeptidase activity in
extracts prepared from magnetically purified
malaria parasites
Fabio L. da Silva, Donald L. Gardiner, and Katharine R. Trenholme
Department of Biology, Queensland Institute of Medical Research, Brisbane, Australia
Introduction
Despite extensive eradication campaigns
malaria remains a major cause of morbidity
and mortality worldwide with an estimated
1–3 million deaths each year¹. The prevention
and treatment of this disease is becoming
increasingly difficult due to the spread of drugresistant parasites and, therefore, there is an
urgent need to discover novel targets for new
drug development.
The aminopeptidases are a group of enzymes
identified within the genome of the most lethal
malaria parasite, Plasmodium falciparum, and
which may have the potential to become new
targets for antimalarial drug development.
These enzymes catalyze the cleavage of the
N-terminal amino acids from proteins and
peptides and one such enzyme, the prolyl
aminopeptidase (PfS33PAP) is currently
being investigated in our laboratory. As a
first step in characterizing the PfS33PAP, it
was necessary to identify the enzyme activity
in extracts of malaria parasites. However, for
biochemical studies it is often crucial to obtain
pure preparations of parasite-infected red
blood cells (RBCs) that are relatively free of
uninfected cells.
P. falciparum passes through a series of
morphologically distinguishable stages as they
mature within RBCs, developing from ring
stages into trophozoites and then into schizontstage parasites. Each parasite contains up to 16
daughter merozoites, which are released into
the blood stream to invade new RBCs. During Purification of P. falciparum–infected
its 48-hour development cycle the parasite erythrocytes using the autoMACS® Pro
digests 65–70% of the host cell hemoglobin Separator
producing insoluble hemozoin as a by- Following the 30-minute incubation in RPMI,
product. The magnetic properties of hemozoin the cells were gently resuspended. The sample
allow the isolation of parasitized erythrocytes. was split into five 10-mL aliquots in 15-mL
Techniques for the in vitro cultivation of P. tubes and kept at 37 °C prior to separation.
falciparum are well established and relatively The first sample was placed in position A1 of
high parasitemia can be achieved, though this a Chill 10 Rack at room temperature. Empty
is dependent on the particular parasite line 15-mL tubes for the collection of the negative
and skill of operator. In this report we present and positive fractions were place in position B1
a protocol for the rapid magnetic purification and C1, respectively. The rack was placed onto
of P. falciparum–infected RBCs from in vitro the MACS® MiniSampler of the autoMACS®
cultures using the autoMACS® Pro Separator. Pro Separator. Cell enrichment was performed
Extracts prepared from purified parasites using program PMalaria. The remaining
exhibited prolyl aminopeptidase activity.
samples were consecutively processed in the
same way. Prior to separation each sample was
gently mixed to resuspend cells.
Preparation of parasite cultures
P. falciparum parasites were cultured and Assessment of yield
synchronized in vitro using established All positive fractions were combined and
methods²,³. An in vitro culture of trophozoite- centrifuged at 400×g for 5 minutes. The
stage parasites at 3–5% parasitemia was supernatant was discarded. Approximately
used for further processing. Ten mL of the 3 μL of the cell pellet was smeared onto a
parasite culture (equivalent to 500 µL packed microscope slide, fixed with methanol and
cell volume) were centrifuged at 400×g for stained with Giemsa solution. Parasitemia was
5 minutes, and the supernatant was discarded. determined microscopically.
Cells were washed twice with pre-warmed
(37 °C) RPMI. Subsequently, the 500-µL cell Prolyl aminopeptidase activity assay
sediment was resuspended in 50 mL RPMI Purified parasites were hypotonically lysed
(equivalent to a 1% hematocrit), and incubated with distilled water to obtain active malarial
in a water bath at 37 °C for 30 minutes.
extract. Enzymatic activity of the prolyl
aminopeptidase, PfS33PAP, in malaria
Vol 15 • 1/2013
MACS & more 31
Report
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32 MACS & more
Vol 15 • 1/2013
Report
Enrichment of P. falciparum–infected RBCs
Cultures of trophozoite stage P. falciparum–
infected RBCs at between 3 and 5% parasitemia
were enriched to give a parasitemia of 80–85%
following one round of enrichment using the
autoMACS Pro Separator (fig. 1).
100
Relative activity (%)
80
60
40
20
H-Pro-NHMec
+ o-phenanthroline
H-Pro-NHMec
H-Leu-NHMec
+ o-phenanthroline
H-Ala-NHMec
+ o-phenanthroline
H-Leu-NHMec
0
H-Ala-NHMec
parasites was determined by addition of extract
aliquots to the fluorogenic substrate H-ProNHMec. Reactions were carried out in 96-well
microtiter plates (100 µL total volume, 60 min
incubation at 37 °C) using a spectrofluorimeter
with excitation at 370 nm and emission at
460 nm. Initial rates were obtained over a range
of substrate concentrations in 50 mM Tris-HCl,
pH 7.5. To ensure the prolyl aminopeptidase
activity was not due to malarial or human
metalloproteases, aliquots of malarial extract
were pre-incubated (10 min, 37 °C) with 2 mM
of the metal chelator o-phenanthroline in
50 mM Tris-HCl and then assayed for activity
against H-Pro-NHMec, H-Ala-NHMec,
H-Leu-NHMec, and H-Glu-NHMec.
To distinguish the PfS33PAP activity from
a second putative P. falciparum prolyl
aminopeptidase, extracts from a PfS33PAP
knockout parasite line were tested for H-ProNHMec activity and compared to 3D7 wild
type parasites. Assays were performed in the
presence of 2 mM phenathroline to inhibit
metalloprotease activity.
Figure 2 H-Pro-NHMec hydrolysis in P. falciparum extracts. Hydrolysis was measured in the presence
or absence of the metal chelator o-phenanthroline (2 mM) as described in the materials and methods section.
Data reflect the mean relative hydrolysis activity (n=2) of 3D7 extract with 200 µM H-Pro-NHMec, 10 µM
H-Ala-NHMec, and 5 µM H-Leu-NHMec as substrates.
P. falciparum extracts contain prolyl
aminopeptidase activity
Soluble extracts of hypotonically lysed wild
type parasites exhibited prolyl aminopeptidase
activity towards H-Pro-NHMec, which
was only partially inhibited by the metal
chelator o-phenanthroline (fig. 2). In contrast,
aminopeptidase activities towards the
substrates H-Ala-NHMec, H-Leu-NHMec
were completely inhibited by the chelator. This
result indicates that at least part of the prolyl
aminopeptidase activity was not due to malarial
or human metalloproteases. To distinguish
between PfS33PAP activity and that of a second
putative malarial prolyl aminopeptidase, we
compared the enzymatic activity in extracts
of wild type and a PfS33PAP knockout strain
and found that o-phenanthroline-insensitive
prolyl aminopeptidase activity was reduced in
the knockout (fig. 3). The remaining activity in
the knockout might be due to a second as yet
uncharacterized prolyl aminopeptidase.
H-Pro-NHMec + o-phenanthroline
Relative activity (%)
100
80
60
40
20
0
Wild type
Figure 1 P. falciparum–infected erythrocytes
isolated using the autoMACS Pro Separator. The
image shows a light-microscopic view of a Giemsastained thin smear of enriched infected cells.
PfS33PAP knockout
Figure 3 H-Pro-NHMec hydrolysis in extracts from 3D7 wild type and PfS33PAP knockout. Prolyl
aminopeptidase activity was measured using extracts prepared from wild type and the PfS33PAP knockout
strain. Tests were performed in the presence of 2 mM o-phenanthroline.
Vol 15 • 1/2013
MACS & more 33
Report
Conclusion
• PfS33 PAP enzyme activity was detected
• The autoMACS® Pro Separator allows the
in extracts prepared from wild type
quick and efficient isolation of P. falciparum–
P. falciparum parasites, and was reduced in
infected RBCs to high purities.
extracts prepared from a transgenic PfS33
• Use of the autoMACS Pro Separator
PAP knockout parasite line.
minimizes manual handling steps during
separation, enabling operator-independent References
1.World Malaria Report 2008. Available at
separation results.
ht t p : / / w w w. w h o. i nt / m a l a r i a / w m r 2 0 0 8 /
• Enzyme activity can be measured in extracts
malaria2008.pdf
prepared from P. falciparum malaria parasites 2. Trager, W. and Jensen, J.B. (1976) Science 193:
673–675.
purified with the autoMACS® Pro Separator.
MACS Product*
Order no.
autoMACS Pro Separator – Starter
Kit
130-092-545
3.Lambros, C. and Vanderberg, J.P. (1979) J.
Parasitol. 65: 418–420.
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Vol 15 • 1/2013
MACS & more 35
Cell separation renewed
Next-generation cell isolation kits
Miltenyi Biotec introduces the next generation
of cell isolation kits. All the reliability you expect.
All the flexibility you need. Now with improved
performance – higher purity, greater recovery, and
faster protocols.
Read the news article on page 5 or visit
www.macscellseparation.com/new-kits
for more information.
Better performance
Exceptional purity and unmatched recovery
Reliable technology
MACS® Technology – the solid foundation for
your research
Faster protocol
Isolate cells in as little as 25 minutes
miltenyibiotec.com
130-100-073
Reliability now comes with improved performance

The path to new discoveries

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