γ α

Transcription

γ α

PRODUCT INFORMATION AND MANUAL
FlowCytomix
Mouse Th1/Th2 10plex Sample Kit
(GM-CSF, IFN-γ, IL-1α, IL-2, IL-4,
IL-5, IL-6, IL-10, IL-17, TNF-α)
BMS820FFSA
For research use only.
Not for diagnostic or therapeutic procedures.
26 Tests (10 analytes)
Mouse Th1/Th2 10plex
BMS820FFSA
Bender MedSystems GmbH
Campus Vienna Biocenter 2
A-1030 Vienna, Austria, Europe
TABLE OF CONTENTS
1
Intended Use
3
2
Summary
4
3
Principles of the Test
5
4
Reagents Provided
7
5
Storage Instructions
9
6
Specimen Collection
9
7
Materials Required But Not Provided
10
8
Precautions for Use
11
9
Preparation of Reagents and Samples
13
10
Technical Tipps
16
11
Test Protocol
18
12
Cytometer Setup
23
13
Calculation of Results
32
14
Limitations
34
15
Performance Characteristics
35
16
FAQ for FlowCytomix Kits
39
17
Bibliography
43
18
Ordering Information
45
19
Reagent Preparation Summary
46
20
Test Protocol Summary
47
BMS820FFSA mouse Th1/Th2 10plex
27.05.09 (06)
3
1 INTENDED USE
BMS820FFSA is a bead based Analyte Detection System for
quantitative detection of mouse Granulocyte Macrophage Colony
Stimulating Factor, Interferon-γ, Interleukin-1α, Interleukin-2,
Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-10, Interleukin-17
and Tumor Necrosis Factor-α by Flow Cytometry. BMS820FFSA is for
research use only. Not for use in diagnostic or therapeutic
procedures.
4
2 SUMMARY
The term TH1 cytokines (referred to also as Type-1 cytokines) and TH2
cytokines (referred to also as Type-2 cytokines) refers to the patterns of
cytokines secreted by two different subpopulations of murine CD4 (+) Tcells that determine the outcome of an antigenic response toward
humoral or cell-mediated immunity.
Numerous cells other than T-cells expressing CD4 have been shown to
be capable of producing TH1 cytokines and TH2 cytokines. These cells
include CD8 (+) T-cells, monocytes, natural killer cells, B-cells,
eosinophils, mast cells, basophils, and other cells.
Type-1 cytokines include IL-2, IFN-gamma, IL-12 and TNF-beta, while
Type-2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13.
Type-1 helper cells (TH1), but not type-2 helper cells (TH2), secrete IL2, IFN-gamma and TNF-beta, whereas TH2 cells, but not TH1 cells,
express IL-4, IL-5, IL-6 and IL-10.
The molecular mechanisms underlying the evolution of these two
different cell types from common precursors are still not completely
known. Studies with transgenic mice carrying null mutations of the IL-4
gene have shown that IL-4 plays an important role in the establishment
of a functional TH2 immune response.
The different patterns of cytokine secretion correspond with different
functions as immune effectors. TH1 cells promote cell-mediated effector
responses. TH2 cells are mainly helper cells that influence B-cell
development and augment humoral responses such as the secretion of
antibodies, predominantly of IgE, by B-cells. Both types of TH cells
influence each other by the Cytokines they secrete; IFN-gamma, for
example, can down regulate TH2 clones while TH2 cytokines, such as
IL-10 can suppress TH1 functions. IFN-gamma has been shown also to
inhibit the proliferation of murine TH2 cells but not that of TH1 helper Tlymphocyte clones. It thus appears that these functional subsets are
mutually antagonistic such that the decision of which subset
predominates within an infection may determine also its outcome.
5
3 PRINCIPLES OF THE TEST
3.1 Principles of the Fluorescent Bead Immunoassay
Beads are coated with antibodies
specifically reacting with each of the
analytes to be detected in the multiplex
system. The beads can be differentiated by
their sizes and by their distinct spectral
addresses.
Fig. 1
Bead with capture antibody
A mixture of coated beads for each analyte
to be measured is incubated with the
samples or standard mixture. The analytes
present in the sample bind to the
antibodies linked to the fluorescent beads.
Fig. 2
A biotin-conjugated second antibody
mixture is added, the specific antibodies
bind to the analytes captured by the first
antibodies.
Fig. 3
- Biotinylated Antibody
Streptavidin - Phycoerythrin is added,
binds to the biotin conjugate and emits
fluorescent signals.
Fig. 4
- Streptavidin-PE
6
3.2 Principles of the FlowCytomix
1023
Fig. 5
SSC-H
Two sets of beads of different size are
used for the FlowCytomix:
Size A: 5.5 µm
Size B: 4.4 µm
R1
0
R2
0
Size A set consists of 11 bead populations,
size B set consists of 9 bead populations
internally dyed with different intensities of a
fluorescent dye (see Fig. 6 and Fig. 7).
Fig. 6
The dye excites with an Argon, He-Ne or
even UV laser, and emits in the far red
(690 nm).
The two different bead sizes make it
possible to distinguish 20 bead sets in one
fluorescence channel.
20 different bead sets distinguished by
internal dye intensity and bead size allow
the simultaneous quantification of 20
analytes in a single small volume sample
using the same principle as an ELISA
(refer to 3.1).
Fig. 7
FSC-H
1023
7
4 REAGENTS PROVIDED
1 vial Setup Beads (SB)
10 vials (175 µl) Fluorescent Beads (20x) coated with specific
antibodies
Table 1
Antibody
Specificity
Bead
Population
Clonality
m GM-CSF
B8
monoclonal
m IFN-γ
B6
monoclonal
m IL-1α
A4
monoclonal
m IL-2
A6
monoclonal
m IL-4
B9
monoclonal
m IL-5
A8
monoclonal
m IL-6
A10
monoclonal
m IL-10
A12
monoclonal
m IL-17
B10
monoclonal
m TNF-α
B7
polyclonal
8
10 vials Standard (lyophilized)
Table 2
Standard
Concentration upon
reconstitution
m GM-CSF
400 ng/ml
m IFN-γ
400 ng/ml
m IL-1α
400 ng/ml
m IL-2
400 ng/ml
m IL-4
400 ng/ml
m IL-5
400 ng/ml
m IL-6
400 ng/ml
m IL-10
400 ng/ml
m IL-17
400 ng/ml
m TNF-α
400 ng/ml
10 vials (100 µl) Biotin-Conjugate (20x) (specific antibody conjugated
to biotin)
Table 3
Biotin-Conjugate
Specificity
Clonality
m GM-CSF
monoclonal
m IFN-γ
polyclonal
m IL-1α
monoclonal
m IL-2
monoclonal
m IL-4
monoclonal
m IL-5
monoclonal
m IL-6
monoclonal
m IL-10
polyclonal
m IL-17
monoclonal
m TNF-α
polyclonal
9
1 bottle (50 ml) Assay Buffer (10x) (PBS with 10% BSA)
1 vial (200 µl) Streptavidin-Phycoerythrin (Streptavidin-PE)
5 STORAGE INSTRUCTIONS
Store kit and components at 2 to 8°C. The expiry of the kit components
can only be guaranteed if the components are stored properly, and if, in
case of repeated use of one component, the reagent is not
contaminated by the first handling.
6 SPECIMEN COLLECTION
Cell culture supernatant and serum were tested with this assay. Other
biological samples might be suitable for use in the assay. Remove
serum from the clot, as soon as possible after clotting.
Samples containing a visible precipitate must be clarified prior to
use in the assay. Do not use grossly lipemic specimens. Fat
causes agglutination of the beads. Centrifugation of lipemic
samples (about 16.000 x g for 5 min) before analysis is
recommended.
Pay attention to a possible “Hook Effect” due to high sample
concentrations (see 15.4.).
Clinical samples should be kept at 2° to 8°C and separated rapidly
before storing at -20°C to avoid loss of bioactivity. If samples are to be
run within 24 hours, they may be stored at 2° to 8°C. Avoid repeated
freeze-thaw cycles.
10
7 MATERIALS REQUIRED BUT NOT PROVIDED
− A flow cytometer equipped with one laser (488 nm or 532 nm)
capable of detecting and distinguishing fluorescence emissions at
575 nm and far red (685 - 690 nm)
− FlowCytomix Pro Software: The FlowCytomix Pro Software is
complimentary and can be ordered at
[email protected] (Cat. No. BMS8400FF) or
downloaded at www.bendermedsystems.com/software-download.
− In the case a filter plate is used, a Filtration manifold is required
(NOTE: The filter plate and adhesive films are not included in the
sample kit, but in regular FlowCytomix Multiplex kits):
We recommend to use the “Multi-Well Plate Vacuum Manifold”
(PALL, cat # P/N 5017) for bead washing.
The Filtration manifold can be ordered via Bender MedSystems in
combination with the FlowCytomix Kit:
Multi-Well Plate Vacuum Manifold, PALL (BMS497FF)
− Centrifuge
− Sample acquisition tubes for a flow cytometer
− Aluminium foil
− 5 ml and 10 ml graduated pipettes
− 10 µl to 1,000 µl adjustable single channel micropipettes with
disposable tips
− 20 µl to 300 µl adjustable multichannel micropipettes with disposable
tips
− Multichannel micropipette reservoir (only required for filter plate
procedure)
− Beakers, flasks, cylinders necessary for preparation of reagents
− Glass-distilled or deionized water
− Vortex mixer
11
8 PRECAUTIONS FOR USE
− All chemicals should be considered as potentially hazardous. We
therefore recommend that this product is handled only by those
persons who have been trained in laboratory techniques and that it is
used in accordance with the principles of good laboratory practice.
Wear suitable protective clothing such as laboratory overalls, safety
glasses and gloves. Care should be taken to avoid contact with skin
or eyes. In the case of contact with skin or eyes wash immediately
with water. See material safety data sheet(s) and/or safety
statement(s) for specific advice (www.bendermedsystems.com).
− Reagents are intended for research use only and are not for use in
diagnostic or therapeutic procedures.
− Do not mix or substitute reagents with those from other lots or other
sources.
− Do not use kit reagents beyond expiration date on label.
− Do not expose kit reagents to light during storage or incubation; the
beads and Streptavidin-Phycoerythrin are photosensitive!
− Do not pipette by mouth.
− Do not eat or smoke in areas where kit reagents or samples are
handled.
− Avoid contact of skin or mucous membranes with kit reagents or
specimens.
− Rubber or disposable latex gloves should be worn while handling kit
reagents or specimens.
− Avoid splashing or generation of aerosols.
− In order to avoid microbial contamination or cross-contamination of
reagents or specimens which may invalidate the test use disposable
pipette tips and/or pipettes.
− Use clean, dedicated reagent trays for dispensing the conjugates.
− Exposure to acids will inactivate the conjugate.
12
− Glass-distilled water or deionized water must be used for reagent
preparation.
− Decontaminate and dispose of specimens and all potentially
contaminated materials as if they could contain infectious agents.
The preferred method of decontamination is autoclaving for a
minimum of 1 hour at 121.5°C.
− Liquid wastes not containing acid and neutralized waste may be
mixed with sodium hypochlorite in volumes such that the final mixture
contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective
decontamination. Liquid waste containing acid must be neutralized
prior to the addition of sodium hypochlorite.
13
9 PREPARATION OF REAGENTS AND SAMPLES
Bring all reagents to room temperature and vortex well before usage!
Calculation of reagent volumes using FlowCytomix Pro Software
Before starting the hands-on work this function facilitates the calculation
of reagent volumes specifically for the number of samples you choose.
Go to “Start/Programs/Bender MedSystems/Test-Setup” to open the
calculator. Enter the number of parameters and the number of your
samples. Choose single or dual evaluation for both standards and
samples. The program automatically provides you with the customized
volumes. Please note that the volumes given by the program are the
exact volumes required in your assay. We therefore recommend to
generously round up the final volumes in order to prepare enough of all
reagents and mixtures.
9.1 Assay Buffer
Mix the contents of the bottle well. Add contents (50.0 ml) Assay Buffer
Concentrate (10x) to 450 ml distilled or deionized water and mix gently
to avoid foaming. Store at 2° to 8°C.
14
9.2 Preparation of Standard
Preparation of Standard Mixture:
Determine the number of analytes.
It is recommended to centrifuge vials for a few seconds in a
microcentrifuge before opening to collect lyophilized standard at the
bottom. The lyophilized standard must be reconstituted by adding
distilled water according to the label on the standard vial. Swirl vial
thoroughly to ensure quantitative solubilization of contents. Wait ten
minutes before pipetting the standard.
It is recommended to centrifuge vials for a few seconds in a
microcentrifuge before pipetting reconstituted standard.
Add 10 µl of each reconstituted standard to a vial labeled standard 1.
Fill up to the final volume of 200 µl with Assay Buffer (1x). (This is a 1:20
dilution of each reconstituted standard.)
Serial Dilution of Standard Mixture:
Add 100 µl Assay Buffer (1x) to 6 tubes labelled standard 2 to 7.
Transfer 50 µl of standard 1 to tube 2, mix the contents of tube 2 and
transfer 50 µl to tube 3. Repeat the procedure creating a row of 7
standard dilutions.
Discard immediately any solubilized or prediluted standard left after
usage.
Fig. 8
15
9.3 Preparation of Bead Mixture
Prepare the Bead Mixture in a centrifuge tube according to the following
calculation.
a. Per test ( = well) 25 µl of the Bead Mixture is required. Consider
tests for standard curves, blanks and samples plus an additional test
for standard 1 for setup.
Calculate the final volume (V fin) of the Bead Mixture needed.
V fin = number of tests x 25 µl
Round up for pipetting reservoir. See also Table 4.
b. Vortex individual bead vial for 5 seconds and pipette 1/20 of final
volume (V fin) of each bead set to a vial labelled “Bead Mix”.
e.g.: V fin = 800 µl; 1/20 of final volume = 40 µl
c.
Fill up to the final volume (V fin) with Assay Buffer (1x).
d. Centrifuge at 3000 x g for 5 min.
e. Carefully remove excess liquid from the surface, leaving 50 µl in the
vial (e.g.: total volume 800 µl, take off 750 µl). Avoid resuspension
of beads.
f.
Add the same volume of Assay Buffer (1x) that has been removed
(e.g.: 750 µl) and vortex for 5 seconds.
Table 4
Number
of tests
26
Volume of
Volume of
each Bead Set Assay Buffer
(µl)
(1x) (µl)
40
400
Final volume of
Bead Mixture
(µl)
800
16
9.4 Preparation of Biotin-Conjugate Mixture
Prepare the Biotin-Conjugate Mixture according to the following
calculation.
a. Per test ( = well) 50 µl of the Biotin-Conjugate Mixture is required.
Consider tests for standard curves, blanks and samples plus an
additional test for standard 1 for setup.
Calculate the final volume (V fin) of the Biotin-Conjugate Mixture
needed.
V fin = number of wells x 50 µl
Round up for pipetting reservoir. See also Table 5
b. Pipette 1/20 of final volume (V fin) of each Biotin-Conjugate to a vial
labelled “Biotin-Conjugate Mix”.
e.g.: V fin = 1600 µl, 1/20 of final volume = 80 µl
c.
Fill up to the final volume (V fin) with Assay Buffer (1x) Dilution.
Table 5
Number
of tests
26
Volume of each
Biotin-Conjugate
(µl)
80
Volume of
Assay Buffer
(1x) (µl)
800
Final volume of
Biotin-Conjugate
Mixture (µl)
1600
9.5 Preparation of Streptavidin-PE Solution
Make a dilution of the concentrated Streptavidin-PE in Assay Buffer (1x)
according to Table 6.
Table 6
Number
of tests
26
conc.
Streptavidin-PE
(µl)
64
Volume of
Assay Buffer
(1x) (µl)
1936
Final volume of
Streptavidin-PE
Solution (µl)
2000
The volumes can be adjusted to the number of tests needed.
17
10 TECHNICAL TIPPS
Please read carefully before you start:
10.1 Mixing
When preparing the Bead Mixture, pipette the bead solution all the way
down to the bottom of the tube, so that you do not lose any material
on the walls of the tube.
Inadequate mixing can lead to little or no beads being detected. Vortex
the beads immediately before mixing with the standards or samples and
again before analysis on the flow cytometer. Vortex each sample tube
for 3-5 seconds before placing the tube on the flow cytometer as this will
yield better discrimination of the bead populations in the FL3/FL4
channel.
10.2 To avoid double bead populations in the analysis plot
When applying the bead solution to the assay plate, put the beads into
the standard- / sample solution in the well to make sure that the
beads do not stick on the side of the tube/well.
(Beads which do not come in contact with the detection antibody during
the incubation step will show a much lower PE signal. These beads will
lead to a second, almost “PE-negative” population.)
10.3 To optimize sensitivity
It is critical that all steps after the addition of the detector reagents are
protected from light to obtain maximum fluorescence of the PE
reporter system.
When establishing the instrument settings with Standard one (highest
concentration), it is essential to place the bead populations at the
very right margin of the acquisition plot. Thereby the distribution of
all standard concentrations across all four decades in FL-2 will be
optimized.
10.4 Blocked filter plate
Serum or plasma samples can sometimes block the filter plate during
washing. If this occurs very carefully push a needle through the hole
underneath the respective well of the plate. Do not stick the needle all
the way through the filter as this will damage the membrane!
18
11 TEST PROTOCOL
11.1 Test Procedure Using the Filter Plate
NOTE: The filter plate and adhesive films are not included in the sample
kit, but in regular FlowCytomix Multiplex kits.
In this case a filtration manifold (see chapter 7) is required for test
performance.
For the FlowCytomix sample kit using tubes please refer to
chapter Fehler! Verweisquelle konnte nicht gefunden werden..
Prepare Assay Buffer referring to 9.1. Prepare Biotin-Conjugate, Bead
Mixtures and standards referring to 9.4, 9.3 and 9.2.
a. Determine the number of microwell strips required to test the desired
number of samples plus appropriate number of wells needed for
running blanks and standards and calculate amount of reagents
needed respectively (see chapter 9). Place adhesive film over
unused wells so that vacuum filtration works efficiently.
We highly recommend to prepare 2 standard curves and an
additional standard 1 (see Table 7 for pipetting scheme) in order
to have enough solution of standard 1 also for cytometer setup
(section 12 of this manual).
b. Add 50 µl Assay Buffer (1x) to the filter plate to pre-wet the wells.
Aspirate using the vacuum filtration manifold. Blot the bottom of the
plate after filtration.
c. Add 25 µl of Standard Mixture dilutions 1 to 7 to designated wells
of the plate (refer to Preparation of Standard 9.2).
19
Table 7
An example of the arrangement of blanks, standards and samples in
the microwell strips:
1
2
3
A
Standard 1
Standard 1
Standard 1
for Setup
B
Standard 2
Standard 2
Sample 1
C
Standard 3
Standard 3
Sample 2
D
Standard 4
Standard 4
Sample 3
E
Standard 5
Standard 5
………..
F
Standard 6
Standard 6
………..
G
Standard 7
Standard 7
………..
H
Blank
Blank
………..
d. Add 25 µl of Assay Buffer (1x) to the blank wells.
e. Add 25 µl of Standard Mixture dilution 1 to well A3 (see Table 7)
which is designated for cytometer setup (highly recommended).
f. Add 25 µl of sample to the designated wells.
g. Add 25 µl of Bead Mixture (refer to Preparation of Bead Mixture 9.3)
to all wells, including the blank wells.
h. Add 50 µl of Biotin-Conjugate Mixture (refer to Preparation of
Biotin-Conjugate Mixture 9.4) to all wells, including the blank wells.
i. Cover wells with adhesive film, avoid putting an excessive pressure
on the top of the plate. Protect from light with an aluminium foil and
incubate at room temperature (18° to 25°C) for 2 hours on a
microplate shaker at 500 rpm.
j. Prepare Streptavidin-PE Solution (refer to Preparation of
Streptavidin-PE 9.5).
20
k. Remove adhesive film and empty wells using the vacuum filtration
manifold. Add 100 µl Assay Buffer (1x) to microwell strips and
empty wells again using the vacuum filtration manifold. Repeat this
step once. Remove any liquid on the bottom of the filter plate with an
absorbent towel. Remove towel before next step.
l. Add 100 µl Assay Buffer (1x) to each well.
m. Add 50 µl of Streptavidin-PE Solution to all wells including the
blank wells.
n. Cover with an adhesive film, avoid putting an excessive pressure on
the top of the plate. Protect from light with an aluminium foil and
incubate at room temperature (18° to 25°C) for 1 hour on a
microplate shaker at 500 rpm.
o. Remove adhesive film and empty wells using the vacuum filtration
manifold. Add 100 µl Assay Buffer (1x) to microwell strips and
empty wells again using the vacuum filtration manifold. Repeat this
step once. Remove any liquid on the bottom of the filter plate with an
absorbent towel. Remove towel before next step.
p. Add 200 µl Assay Buffer (1x) to each well.
q. Mix the contents of each well by repeated aspiration and ejection,
and transfer these 200 µl of each well into a separate sample
acquisition tube for a flow cytometer and fill up to 500 µl final volume
by adding 300 µl of Assay Buffer (1x).
r. Proceed to next step of protocol or store tubes for a maximum of
24 hours at 2 – 8°C protected from light. Swirl tube contents
thoroughly if they are stored longer than 2 hours.
s. Before analysing samples on a flow cytometer make cytometer
setup (refer to chapter 12).
21
11.2 Test Procedure Using Tubes
In this case incubations are performed in tubes. Centrifugation steps are
necessary for test performance.
Prepare Assay Buffer referring to 9.1. Prepare Biotin-Conjugate, Bead
Mixtures and standards referring to 9.4, 9.3 and 9.2.
a. Determine the number of cytometer tubes required to test the desired
number of samples plus appropriate number of tubes needed for
running blanks and standards and calculate amount of reagents
needed respectively (see chapter 9).
We highly recommend to prepare 2 standard curves and an
additional standard 1 (see Table 7 for pipetting scheme) in order
to have enough solution of standard 1 also for cytometer setup
(section 12 of this manual).
b. Add 25 µl of Standard Mixture dilutions 1 to 7 in designated tubes
(refer to Preparation of Standard 9.2).
c. Add 25 µl of Assay Buffer (1x) to the blank tubes.
d. Add 25 µl of Standard Mixture dilution 1 to tube 17 which is
designated for cytometer setup (highly recommended).
e. Add 25 µl of sample to the designated sample tubes.
f. Add 25 µl of Bead Mixture (refer to Preparation of Bead Mixture 9.3)
to all tubes, including the blank tubes.
g. Add 50 µl of Biotin-Conjugate Mixture (refer to Preparation of
Biotin-Conjugate Mixture 9.4) to all tubes, including the blank tubes.
h. Mix the contents of each tube well and incubate at room temperature
(18° to 25°C) for 2 hours. Protect from light with an aluminium foil.
i. Prepare Streptavidin-PE Solution (refer to Preparation of
Streptavidin-PE 9.5).
j. Add 1 ml of Assay Buffer (1x) to all tubes and spin down at
200 x g for 5 minutes.
22
k. Carefully discard the supernatant from each tube leaving 100 µl of
liquid in each tube.
l. Repeat steps j. and k.
m. Add 50 µl of Streptavidin-PE Solution to all tubes including the
blank tubes.
n. Mix the contents of each tube well and incubate at room temperature
(18° to 25°C) for 1 hour. Protect from light with an aluminium foil.
o. Add 1 ml of Assay Buffer (1x) to all tubes and spin down at 200 x g
for 5 minutes.
p. Carefully discard the supernatant from each tube leaving 100 µl of
liquid in each tube.
q. Repeat steps o and p.
r. Add 500 µl Assay Buffer (1x) to each tube.
s. Proceed to next step of protocol or store tubes for a maximum of
24 h at 2 – 8°C protected from light. Swirl tube contents thoroughly if
they are stored longer than 2 hours.
t. Before analysing samples on a flow cytometer make cytometer
setup (refer to chapter 12).
23
12 CYTOMETER SETUP
12.1 Preparation of Cytometer Setup Beads
Vortex the vial with Setup Beads for several seconds. Pipette 500 µl of
Setup Beads into a tube labelled SB.
12.2 Instrument Setup
Please note before setup:
The Setup Beads provided with this assay are needed
• to adjust FS/SSC parameters
• to create regions for the bead populations
• to define the number of events counted
• to adjust voltage of FL-2 so that the bead populations are positioned
in the left part of the dot blot. This ensures that the bead populations
of standard 1 will be visible on the screen.
With every new experiment before starting sample acquisition, adjust
voltage of FL-2/FL-3(-4) and compensation with standard 1 in “Setup
Mode” (prepared twice for this purpose, see 11), which gives the highest
PE (FL-2) signal.
After all settings and compensation have been optimized, switch to
“Acquisition Mode” and start measuring standards and samples.
• Do not change Voltage and Compensation, nor Flow Through
(small – medium – high) during measurement.
For the most commonly used flow cytometers (e.g.Becton Dickinson or
DakoCytomation) you can immediately start cytometer setup as below.
If you run a FlowCytomix assay on a FC500 Instrument from
Beckman Coulter you must ensure that the Forward Scatter (FS)
measurements are collected at 1-8°. To accomplish this you must insert
the FS 1-8° Field Stop into place: remove the front cover to locate the
FS 1-8° Field Stop. Slide the knob from the right (= default 1-19°
position) to the left and push to lock in place.
On the XL Series systems the EPICS XL/XL-MCL FS Low Angle
Collection Kit is recommended to accomplish this.
Then proceed to general protocol.
24
a. Perform instrument start up following the manufacturer´s
recommendations.
b. Perform flow check to verify alignment and fluidic stability of the
system.
c. Open a new protocol.
d. Create a Dot Plot window with FS (Forward Scatter) for X- and SSC
(Side Scatter) for Y-axis.
e. Set FS and SSC to linear mode.
f. Create a second and a third Dot Plot window with FL-2 for X- and FL3 (-4) for Y-axis.
NOTE:
The FL channel number for the specific nm range depends on
the instrument. e.g.: 690 nm is FL-3 in a BD FACSCalibur but
FL- 4 in a Beckman Coulter Flow Cytometer
g. Set FL-2 and FL-3 (-4) to Log mode.
h. Set Compensation to zero.
i. Save your protocol frequently during setup.
j. In “Setup Mode” run tube SB (with Setup Beads).
k. Adjust the parameters of FS and SSC so that both bead populations
in the control are visible in the opened window (see Fig. 9).
Fig. 9
(Pause and restart acquisition
frequently during the cytometer
setup in order to reset detected
values after setting adjustment.)
25
l. Create regions “R1” for the large beads and “R2” for the small beads
(see Fig. 10).
Fig. 10
Define number of events so that 300 events per analyte are
measured within gate R2 (small beads) e.g. count 1500 events of
gated bead population R2 for the 10-plex (300 events per analyte –5
small bead populations >> 1500 events).
26
m. Select gate R1 in the first FL-2/FL-3(-4) Dot Plot (Fig. 11, Gate R1).
Select gate R2 in the second FL-2/FL-3(-4) Dot Plot (see Fig. 11,
Gate R2).
Fig. 11
Gate R1
Gate R2
Adjust the parameters of FL-2 and FL-3 (-4) so that the bead
populations are visible in the opened window.
With Setup Beads of tube SB voltage of FL-2 has to be adjusted so
that the bead populations are positioned in the left part of the dot
blot. This ensures that the bead populations of standard 1 will be
visible on the screen.
n. Save your protocol frequently during setup.
27
o. Before starting the acquisition of standards and samples stay in
SETUP MODE and adjust the settings using standard 1, which gives
the highest PE (FL-2) signal.
Adjust the parameters of FL-2 so that the bead population with the
highest PE (FL-2) signal sticks to the right axis (in order to
guarantee that bead populations with low PE/low concentrated
analytes are detectable) while the whole population is visible (see
Fig. 12).
Fig. 12
Gate R1
Gate R2
IL-10
IL-6
IL-5
IL-2
IL-17
IL-4
GM-CSF
TNF-α
IFN-γ
IL-1α
p. Restart acquisition of standard 1 for setup.
(Pause and restart acquisition frequently during the cytometer setup
in order to reset detected values after setting adjustment)
28
q. If the bead populations are not in a horizontal position (as shown in
Fig. 13) increase compensation setting for FL-3 (-4) - %FL-2.
Fig. 13
Gate R1
Gate R2
FL-3(-4) - %FL2
compensation
29
r. If the bead populations are not in a horizontal position (see Fig. 14)
decrease compensation setting for FL-3 (-4) - %FL-2.
Fig. 14
Gate R1
Gate R2
FL-3(-4) - %FL2
compensation
A final setup may look like that shown in section 12.3 of this manual.
s. Save your protocol.
30
12.3 Acquisition
a. Switch from SETUP MODE to ACQUISITION MODE.
b. To simplify auto file loading in the analysis software save all
measured data with the same file name and consecutive numbering
(e.g.: FBI.001, FBI.002, FBI.003, …).
c. Begin analysing samples starting with the standard curve (S1-S7 and
blank), followed by the samples.
A final setup (e.g.: for BD FACSCalibur) may look like that shown in
Table 8:
N.B.: individual setup is required
Table 8
Detector
FS
SSC
FL1
FL2
FL3
FL1
FL2
FL2
FL3
Voltage
E00
350
600
650
610
Compensation
0.0
0.0
0.0
7.5
Amp
5.4
2
1
-
FL2
FL1
FL3
FL2
Mode
Lin
Lin
Lin
Log
Log
31
A final setup (e.g.: for BD FACScan) may look like that shown in
Table 9:
Table 9
Detector
FS
SSC
FL1
FL2
FL3
FL1
FL2
FL2
FL3
Voltage
E01
336
163
316
501
Compensation
0.0
0.0
0.0
10.0
Amp
1.9
1
-
Mode
Lin
Lin
Log
Log
Log
FL2
FL1
FL3
FL2
A final setup (e.g.: for BC FC500) may look like that shown in
Table 10:
Table 10
FS
SSC
FL1
FL2
FL3
FL4
FL5
Detectors
200-400
200-400
200-400
500
250
580
250
Compensation
FL1
FL1
FL2
FL3
FL4
FL5
Gain
20
5 to 10
1
1
1
1
1
FL2
10
32
13 CALCULATION OF RESULTS
For calculation of results refer to the BMS FlowCytomix Software
manual.
BMS FlowCytomix Pro software and manual is included in the kit, but
also available for free download on: www.bendermedsystems.com
Table 11
Representative standard curves.
Do not use these curves to derive test results. A standard curve must be
run for each group of samples assayed.
Concentration
pg/ml
m IL-10
Fluorescent Intensity
m IL-6
m IL-5
m IL-2
m IL-1α
α
20000
91.1
717.8
535.9
201.9
668.8
6667
35.9
325.3
337.2
101.0
343.3
2222
14.5
117.0
163.5
31.2
125.7
741
6.8
38.2
50.9
8.6
32.4
247
3.8
14.7
14.2
3.1
8.0
82
2.5
7.1
4.8
1.6
2.1
27
2.0
4.5
2.6
1.2
1.1
0
1.6
2.7
1.7
0.9
0.8
Concentration
pg/ml
20000
512.3
Fluorescent Intensity
m IL-4 m GM-CSF m TNF-α
α m IFN-γγ
84.4
817.9
346.4
39.7
6667
251.7
367.3
201.9
54.3
20.9
2222
87.9
129.1
68.3
29.1
8.1
741
28.7
39.8
16.8
13.0
3.1
247
10.3
13.7
4.5
5.7
1.3
82
4.3
5.3
1.7
2.6
0.6
27
2.5
2.8
1.1
1.4
0.4
0
1.3
1.2
0.9
0.6
0.3
m IL-17
33
− To determine the concentration of circulating analytes for each
sample, first find the fluorescent intensity value on the ordinate and
extend a horizontal line to the standard curve. At the point of
intersection, extend a vertical line to the abscissa and read the
corresponding concentration.
− It is suggested that each testing facility establishes a control sample
of known concentration and runs this additional control with each
assay. If the values obtained are not within the expected range of this
control, the assay results may be invalid.
34
14 LIMITATIONS
− Since exact conditions may vary from assay to assay, standard
curves must be established for every run.
− Bacterial or fungal contamination of either samples or reagents or
cross-contamination between reagents may cause erroneous results.
− Disposable pipette tips, flasks or glassware are preferred, reusable
glassware must be washed and thoroughly rinsed of all detergents
before use.
35
15 PERFORMANCE CHARACTERISTICS
15.1 Sensitivity
The limit of detection of each analyte defined as the analyte
concentration resulting in a fluorescent intensity significantly higher than
that of the dilution medium (mean + 2 standard deviations) was
determined to be:
Table 12
Analyte
Sensitivity
(pg/ml)
m GM-CSF
10.9
m IFN-γ
6.5
m IL-1α
15.7
m IL-2
8.8
m IL-4
0.7
m IL-5
4.0
m IL-6
2.2
m IL-10
5.4
m IL-17
2.4
m TNF-α
2.1
36
15.2 Reproducibility
15.2.1 Intra-assay
Reproducibility within the assay was evaluated in 3 independent
experiments. Each assay was carried out with 6 replicates of 4 serum
samples containing different concentrations of all analytes. Two
standard curves were run with each assay. Data below show the mean
intra-assay coefficient of variation for each analyte (see Table 13).
Individual user data may vary due to differences in protein content of
serum pools or individual donor serum.
Table 13
The coefficient of variation of the analyte concentration calculated for
each sample.
CV
Sample 1
high (%)
CV
Sample 2
medium
high (%)
CV
Sample 3
medium
low (%)
CV
Sample 4
low (%)
Mean
intraassay
CV (%)
m GM-CSF
1.7
2.9
2.2
5.2
3.0
m IFN-γγ
9.4
2.3
3.9
7.3
5.7
m IL-1α
α
5.6
5.5
1.8
8.8
5.4
m IL-2
4.0
2.1
7.2
6.1
4.9
m IL-4
4.0
3.8
6.1
6.2
5.0
m IL-5
4.0
4.7
2.7
3.0
3.6
m IL-6
2.3
2.5
3.0
7.3
3.8
m IL-10
4.9
3.6
3.5
3.2
3.8
m IL-17
2.0
2.1
3.4
3.8
2.8
m TNF-α
α
6.2
3.2
3.7
3.8
4.2
37
15.2.2 Inter-assay
Assay to assay reproducibility within one laboratory was evaluated in 3
independent experiments. Each assay was carried out with 6 replicates
of 4 serum samples containing different concentrations of all analytes.
Two standard curves were run with each assay. Data below show the
mean inter-assay coefficient of variation for each analyte (see Table 14).
Individual user data may vary due to differences in protein content of
serum pools or individual donor serum.
Table 14
The coefficient of variation of the analyte concentration calculated for
each sample.
CV
Sample 1
high (%)
CV
Sample 2
medium
high (%)
CV
Sample 3
medium
low (%)
CV
Sample 4
low (%)
Mean
interassay
CV (%)
m GM-CSF
6.0
6.6
5.5
17.8
9.0
m IFN-γγ
10.8
5.9
3.9
6.1
6.7
m IL-1α
α
6.6
0.6
3.1
8.0
4.6
m IL-2
6.6
6.6
2.0
15.1
7.6
m IL-4
2.7
2.9
1.6
12.6
5.0
m IL-5
5.9
9.0
8.3
14.4
9.4
m IL-6
11.2
13.8
13.3
5.4
10.9
m IL-10
4.1
6.3
8.3
10.4
7.3
m IL-17
2.0
2.4
4.3
16.3
6.3
m TNF-α
α
2.9
1.8
3.7
3.3
2.9
38
15.3 Specificity
There was no detectable crossreactivity observed for other combinable
analytes of Simplex and Multiplex Assays from Bender MedSystems.
(For detailed information refer to “Combination Table“ on
www.bendermedsystems.com.)
15.4 Hook effect
Samples with expected concentrations two fold higher than the standard
of the highest concentration should be diluted 10 fold in Assay Buffer
(1x) before assay performance to prevent false negative results due to
“hook effects”.
39
16 FAQ FOR FLOWCYTOMIX KITS
Is there a particular flow cytometer required for the use of
FlowCytomix Kits?
The FlowCytomix beads are detectable in the most commonly used flow
cytometers. The flow cytometer needs to be equipped with one laser
(488 nm or 532 nm) capable of detecting and distinguishing
fluorescence emissions at 575 nm and far red (685 nm - 690 nm).
FlowCytomix suitability has been tested for:
Beckman Coulter:
EPICS® XLTM / XL-MCL TM
Cytomics TM FC500
(see section 12.2 of this booklet)
(to resolve the two bead populations, the
Forward Scatter collection angles have to be
at the correct position. Move the Field Stop
mechanism to the left position in order to
change the Forward Scatter collection angles
from 1-19 degrees to 1-8 degrees.)
BD FACScan TM
BD FACSCalibur TM
BD FACSCanto TM
BD TM LSR I
BD TM LSR II
Is there a special software required?
Yes, the FACS raw data is analysed with the specifically designed
FlowCytomixPro Software. The software is included in the FlowCytomix
Multiplex Kits and the FlowCytomix Basic Kits and can be downloaded
from the web page: www.bendermedsystems.com
Does the software interfere with the flow cytometer operating
system?
Software has been virus checked with latest version of McAfee
VirusScan. FlowCytomixPro Software has been successfully operated in
conjunction with all commonly used flow cytometer models. It does not
interfere with the flow cytometer operating system.
40
In addition the FlowCytomixPro Software does not need to be installed
on the flow cytometer working station; data files can be transferred to
any other computer.
What happens if less than 10 analytes have to be measured?
Simplex Kits are available for this purpose. Simplex Kits are designed
for the detection of one specific analyte. Simplex Kits can be combined
to give any combination (within the human or mouse range) and number
of analytes to be measured.
In which case is a Basic Kit required?
A Basic Kit is required for any combination of Simplex Kits. In order to
run a FlowCytomix assay some reagents are needed only once, even in
the case of combining several Simplex Kits. These reagents are
provided in a Basic Kit.
Are the assays performed in plates or in tubes?
The FlowCytomix assays can be either performed like an ELISA in a 96well filter plate, in this case a filtration manifold is required, or
alternatively in tubes, which requires centrifugation of the tubes during
the washing procedure.
Why is a cytometer setup required before measuring standards and
samples?
The cytometer setup is required by the mode of operation of the flow
cytometer. Setup Beads are included in the Basic Kits and in the
Multiplex Kits.
With every new experiment, before starting the acquisition of standards
and samples stay in SET UP MODE and adjust the settings using the
highest standard 1.
What wavelength is chosen for measurement?
The maximum emission of PE is at 578 nm and the emission of the
beads is in the far red region (685 - 690 nm).
Is it possible to measure with high “flow through” settings?
There are three flow through settings: low-medium-high.
Increasing the “flow through” may result in a dispersed bead population.
Therefore it is recommended to start with the low “flow through”; it can
only be increased if the bead population does not start to disperse.
41
Once the final instrument settings are saved, do not change the “flow
through” during measurement.
Does a FlowCytomix Kit work on a LuminexTM instrument?
No, the FlowCytomix Kit can only be measured on a flow cytometer.
Is it correct to use the non linear part of the standard curve?
Yes, it is possible to use the non linear part of the standard curve for
calculation of results. Dilutions of samples behave in the same way as
the standard curve.
FAQ´s regarding Combination:
How many analytes can be combined in one assay?
Up to 20 analytes are possible.
Which analytes can be combined?
There are 20 different bead sets that can be distinguished in the
FlowCytomix assay. Analytes containing different bead sets can be
analysed simultaneously. You can find the analytes and the
corresponding bead set listed at “4 Reagents Provided”.
Can Multiplex Kits be combined with each other?
Multiplex Kits can be combined with each other as long as they do not
use the same bead sets. Pay attention to the kind of Conjugate (PE or
Biotin). For detailed information about possible combinations refer to
“Combination Table“ on www.bendermedsystems.com.
Can Simplex Kits be combined with a Multiplex Kit?
It is possible in case all analytes contain different bead sets. Pay
attention to the kind of Conjugate (PE or Biotin). For detailed information
about possible combinations refer to “Combination Table“ on
www.bendermedsystems.com.
Can components from different lots be mixed?
The bead sets, standards and conjugates are lot-specific and must be
used in combination with each other. Do not mix these components from
different kit lots.
Assay Buffer and Streptavidin-PE are not lot-specific and can therefore
be exchanged between different kit lots.
42
Is it possible to combine Biotin Conjugates with PE-Conjugates in
one Conjugate Mixture?
In case you are using at least one Biotin-Conjugate in your combination
a further incubation step with Streptavidin-PE is necessary. This does
not interfere with the PE-Conjugates.
43
17 BIBLIOGRAPHY
1) Bloom BR et al. Revisiting and revising T suppressor cells.
Immunology Today 13: 131-6 (1992).
2) Bottomly K. A functional dichotomy in CD4 + T lymphocytes.
3) Cherwinski HM et al. Two types of mouse helper T cell clone. III.
Further differences in lymphokine synthesis between Th1 and Th2
clones revealed by RNA hybridization, functionally monospecific
bioassays, and monoclonal antibodies. Journal of Experimental
Medicine 166: 1229-44 (1987).
4) Clerici M and Shearer GM A. Th1 to Th2 switch is a critical step in
the etiology oh HIV infections. Immunology Today 14: 107-111
(1993).
5) Del Prete GF et al. Purified protein derivative of Mycobacterium
tuberculosis and excretory-secretory antigen(s) of Toxocara canis
expand in vitro human T cells with stable and opposite (type 1 T
helper or type 2 T helper) profile of cytokine production. Journal of
Clinical Investigation 88: 346-50 (1991).
6) Fiorentino DF et al. IL10 acts on the antigen-presenting cell to
inhibit cytokine production by Th1 cells. Journal of Immunology
146: 3444-51 (1991).
7) Firestein GS et al. A new murine CD4 + T cell subset with an
unrestricted cytokine profile. Journal of Immunology 143: 518-25
(1989).
8) Gajewski TF and Fitch FW. Anti-proliferative effect of IFN-gamma
in immune regulation. I. IFN-gamma inhibits the proliferation of Th2
but not Th1 murine helper T lymphocyte clones. Journal of
Immunology 140: 4245-52 (1988).
9) Hikida M et al. Suppression of interleukin 4 production from type 2
helper T cell clone by antisense oligodeoxynucleotide. Immunology
Letters 34: 297-302 (1992).
10) Kamogawa Y et al. The relationship of IL4 and IFN-gammaproducing T cells studied by lineage ablation of IL4 producing cells.
Cell 75: 985-95 (1993).
44
11) Kawakami K and Parker DC. Differences between T helper cell
lines in signaling pathways for induction of contact-dependent T
cell help. European Journal of Immunology 22: 85-93 (1992).
12) Kelso A et al. Heterogeneity in lymphokine profiles of CD4 + and
CD8 + T cells and clones activated in vivo and in vitro.
Immunological Reviews 123: 85-114 (1991).
13) Lederer JA et al. Regulation of cytokine gene expression in T
helper cell subsets. Journal of Immunology 152: 77-86 (1994).
14) Mosmann TR et al. Two types of murine helper T cell clone. I.
Definition according to profiles of lymphokine activities and
secreted proteins. Journal of Immunology 136: 2348-57 (1986).
15) Mosmann TR and Coffman RL. Heterogeneity of cytokine secretion
patterns and functions of helper T cells. Advances in Immunology
111-47 (1989).
16) Mosmann TR and Coffman RL. TH1 and TH2 cells: different
patterns of lymphokine secretion lead to different functional
properties. Annual Review of Immunology 7: 145-73 (1989).
17) Paul WE and Seder RA. Lymphocyte responses and cytokines.
Cell 76: 241-51 (1994).
18) Rocken M et al. A common precursor for CD4 + T cells producing
IL2 or IL4. Journal of Immunology 148: 1031-6 (1992).
19) Romagnani S. Type 1 T helper and type 2 T helper cells: functions,
regulation and role in protection and disease. International Journal
of Clin. Lab. Research 21: 152-8 (1991).
20) Romagnani S. Human TH1 and TH2 subsets: doubt no more.
21) Rooney JW et al. A common factor regulates both Th1- and Th2specific cytokine gene expression. EMBO Journal 13:625-33
(1994).
22) Williams ME et al. Activation of functionally distinct subsets of CD4
+ T lymphocytes. Research Immunology 142: 23-8 (1991).
For literature update refer to www.bendermedsystems.com
45
18 ORDERING INFORMATION
For orders please contact:
Europe-Headquarters
Bender MedSystems GmbH
Campus Vienna Biocenter 2
A-1030 Vienna, Austria, Europe
phone: +43 1 796 40 40 ext. 114
fax: +43 1 796 40 40 ext. 400
e-mail: [email protected]
USA
Bender MedSystems, Inc.
849 Hinckley Road
Burlingame, CA 94010, USA
toll-free phone:
+1 (866) 952 2112
toll-free fax:
+1 (877) 952 2112
phone:
+1 (650) 952 2112
fax:
+1 (650) 952 2252
For technical information please contact:
www.bendermedsystems.com
Cat.No. BMS820FFSA mouse Th1/Th2 10plex
46
19 REAGENT PREPARATION SUMMARY
19.1 Assay Buffer
Table 15
Assay Buffer
Concentrate (10x)
(ml)
50
Distilled Water
(ml)
450
19.2 Biotin-Conjugate Mixture
Make a 1:20 dilution of each Biotin-Conjugate creating a Mixture of
Conjugates.
19.3 Bead Mixture
Make a 1:20 dilution of each bead set creating a mixture of beads. Wash
Bead Mixture once with Assay Buffer (1x).
19.4 Standard
a. Centrifuge vials for a few seconds to collect lyophilized standard at
the bottom. Reconstitute the standard by adding distilled water
according to the label on the standard vial.
b. Make a 1:20 dilution of the reconstituted standards all diluted in the
same vial to get standard 1. Make further serial dilutions.
19.5 Streptavidin-PE Solution
Table 16
Number of
tests
26
conc.
Assay Buffer (1x)
Streptavidin-PE
(µl)
(µl)
64
1936
Final volume of
Streptavidin-PE
Solution
(µl)
2000
47
20 TEST PROTOCOL SUMMARY
20.1 USING THE FILTER PLATE: vacuum manifold required
− Prepare Assay Buffer
− Prepare Biotin-Conjugate Mixture, Bead Mixture and Standard
Mixture
− Pre-wet microwell strips of filter plate with Assay Buffer (1x)
− Pipette 25 µl diluted Standard Mixture dilution 1-7 into designated
wells
− Add 25 µl Assay Buffer (1x) to the blank wells
− Add 25 µl of Standard Mixture dilution 1 to well A3 which is
designated for cytometer setup (highly recommended)
− Add 25 µl sample to designated wells
− Add 25 µl Bead Mixture to all wells
− Add 50 µl of Biotin-Conjugate Mixture to all wells
− Cover microwell strips protect from light and incubate 2 hours at
room temperature (18° to 25°C) on microplate shaker (500 rpm)
− Prepare Streptavidin-PE Solution
− Wash microwell strips twice with Assay Buffer (1x) using the
vacuum filtration manifold
− Add 100 µl of Assay Buffer (1x)
− Add 50 µl of Streptavidin-PE Solution to all wells
− Cover microwell strips, protect from light and incubate 1 hour at room
temperature (18° to 25°C) on microplate shaker (500 rpm)
− Wash microwell strips twice with Assay Buffer (1x) using the
vacuum filtration manifold
− Add 200 µl of Assay Buffer (1x)
48
− Mix the contents of each well by repeated aspiration and ejection,
and transfer these 200 µl of each well into a separate sample
acquisition tube for a flow cytometer. Fill up to 500 µl final volume
with Assay Buffer (1x)
− Analyse samples on a flow cytometer.
20.2 USING TUBES: centrifugation steps required
− Prepare Assay Buffer
− Prepare Biotin-Conjugate Mixture, Bead Mixture and Standard
Mixture
− Pipette 25 µl diluted Standard Mixture dilution 1-7 into designated
tubes
− Add 25 µl Assay Buffer (1x) to the blank tubes
− Add 25 µl of Standard Mixture dilution 1 to the tube which is
designated for cytometer setup (highly recommended)
− Add 25 µl sample to designated tubes
− Add 25 µl Bead Mixture to all tubes
− Add 50 µl of Biotin-Conjugate Mixture to all tubes
− Incubate 2 hours at room temperature (18° to 25°C) protect from light
− Prepare Streptavidin-PE Solution
− Add 1 ml Assay Buffer (1x) to all tubes
− Wash tubes twice by centrifugation (5 min at 200 x g) and discard
supernatant carefully leaving 100 µl of liquid in each tube
− Add 50 µl of Streptavidin-PE Solution to all tubes
− Incubate 1 hour at room temperature (18° to 25°C) protect from light
− Add 1 ml Assay Buffer (1x) to all tubes
49
− Wash tubes twice by centrifugation (5 min at 200 x g) and discard
supernatant carefully leaving 100 µl of liquid in each tube
− Add 500 µl Assay Buffer (1x) to all tubes
− Analyse samples on a flow cytometer

γ α

Transcription

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