Thermo Fisher Scientific eBioscience Immune Response T Cell Cytokines Kit User guide

Brand
Thermo Fisher Scientific
Model
eBioscience Immune Response T Cell Cytokines Kit
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
eBioscience™ Immune Response T Cell
Cytokines Kit
USER GUIDE
Catalog Number A53421
Document Part Number 2162761
Publication Number MAN0026418
Revision A.0
Pierce Biotechnology, Inc. | Thermo Fisher Scientific | 3747 N. Meridian Road | Rockford, Illinois 61101 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0026418
Revision Date Description
A.0 24 February 2022 New document created for eBioscience™ Immune Response T Cell Cytokines Kit.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2022 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information .................................................. 4
Product description ............................................................. 4
Contents and storage ............................................................ 4
Required materials not supplied ................................................... 5
■CHAPTER 2 Methods ............................................................... 6
General guidelines .............................................................. 6
Test tube protocol ............................................................... 6
Stimulate cells .............................................................. 6
Stain cells with viability dye .................................................. 7
Fix and permeabilize cells .................................................... 7
Stain cells and compensation beads with fluorophore-conjugated antibodies ....... 8
■APPENDIX A Troubleshooting .................................................... 10
■APPENDIX B 96–well plate protocol ............................................. 11
Stimulate cells ................................................................. 11
Stain cells with viability dye ..................................................... 11
Fix and permeabilize cells ....................................................... 12
Stain cells and compensation beads with fluorophore-conjugated antibodies .......... 12
■APPENDIX C Typical experimental set up and results ......................... 15
Typical experimental set up ...................................................... 15
Typical experimental results ..................................................... 19
■APPENDIX D Safety ............................................................... 21
Chemical safety ................................................................ 21
Biological hazard safety ......................................................... 22
■APPENDIX E Documentation and support ...................................... 23
Customer and technical support ................................................. 23
Limited product warranty ........................................................ 23
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 3
Product information
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The eBioscience™ Immune Response T Cell Cytokines Kit includes all the components needed to
stimulate and detect production of interferon gamma (IFN-γ) and interleukin 17A (IL-17A) from human T
cells by flow cytometry.
Contents and storage
Unless otherwise indicated, all materials are available through thermofisher.com. Catalog numbers
that appear as links open the web pages for those products.
Table 1 eBioscience™ Immune Response T Cell Cytokines Kit (Cat. No. A53421)
Contents Cat. No. Amount Storage
eBioscience™ Phytohemagglutinin-L (PHA-L)
Solution (500X)
00-4977-93 100 μL −20°C
eBioscience™ Protein Transport Inhibitor
Cocktail (500X)
00-4980-93 100 μL
LIVE/DEAD™ Fixable Near IR (780) Viability kit,
for 633 nm excitation
L34993 200 assays
eBioscience™ Flow Cytometry Staining Buer 00-4222-57 200 mL 4°C
eBioscience™ IC Fixation Buer 00-8222-49 125 mL
eBioscience™ Permeabilization Buer (10X) 00-8333-56 100 mL
CD3 Monoclonal Antibody (SK7), APC,
eBioscience™
17-0036-42 100 tests
CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-
eFluor 710, eBioscience™
46-0047-42 100 tests
CD8a Monoclonal Antibody (SK1), eFluor 450,
eBioscience™
48-0087-42 100 tests
1
4eBioscience™ Immune Response T Cell Cytokines Kit User Guide
Table 1 eBioscience Immune Response T Cell Cytokines Kit (Cat. No. A53421) (continued)
Contents Cat. No. Amount Storage
IFN gamma Monoclonal Antibody (4S.B3), FITC,
eBioscience™
4°C11-7319-82 100 μg
IL-17A Monoclonal Antibody (eBio64DEC17),
PE, eBioscience™
12-7179-42 100 tests
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1),
FITC, eBioscience™
11-4714-81 50 μg
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1),
PE, eBioscience™
12-4714-81 50 μg
UltraComp eBeads™ Plus Compensation Beads 01-3333-42 100 tests
Required materials not supplied
•Phosphate-buered saline (PBS), no azide
• Complete tissue culture media
• Distilled water
• Tissue culture plates or flasks
• 12 × 75 mm round bottom test tubes
• (Optional) Peptides or other T cell stimulation reagents of choice
• (Optional) 96-well round- or v-bottom plate
• Flow cytometer equipped with violet, blue, yellow, and red lasers
•Humidified incubator set to 37°C and 5% CO2
• Centrifuge compatible with 12 × 75 mm round bottom test tubes (or 96–well plates)
• Vortex mixer
• Pipettes
Chapter 1 Product information
Required materials not supplied 1
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 5
Methods
General guidelines
•This protocol has been developed using peripheral blood mononuclear cells (PBMC) from normal,
healthy donors. The stimulation conditions (cell concentration, incubation time, and reagent
concentrations) may be further optimized for dierent sample types and alternative stimulation
reagents, such as peptides.
•In this protocol, the stimulations are performed in bulk, then cells are transferred into 12 × 75 mm
round bottom test tubes for staining. To perform the protocol in 96–well plates, see Appendix B,
96–well plate protocol.
•For typical experimental setup, results, and how to gate and analyze data generated with this kit,
see Appendix C, Typical experimental set up and results. An ideal experiment contains:
– Single-color compensation controls (prepared with a combination of the included antibodies,
UltraComp eBeads™ Plus Compensation Beads, and cells of interest)
– Isotype control samples (samples stained with viability dye, T cell phenotyping markers, plus
isotype control antibodies in place of IFN-γ and IL-17A)
– Fully-stained samples (experimental samples stained with viability dye and all target-specific
antibodies)
– Unstimulated, vehicle controls from the same donors, stained with the mixed antibody, isotype
control panel, and the full antibody panel.
Test tube protocol
This protocol is intended for 12 × 75 mm round bottom test tubes. For the 96-well plate protocol, see
Appendix B, 96–well plate protocol.
Stimulate cells
1. Resuspend cells at 2 x 107 cells/mL in complete media.
2. Prepare a 2X working solution of PHA-L by adding 10–20 μL of PHA-L (500X) per 1 mL of complete
media. You will need the same volume as the cell suspension prepared in step 1.
Alternatively, prepare a 2X working solution of a peptide(s) or other stimulation reagent of choice.
Note: This dilution uses PHA-L at a final concentration of 5–10X. Using lower concentrations of
PHA-L may be desired and should be determined empirically.
3. Mix cells and the 2X PHA-L (or other stimulation reagent) at 1:1, then transfer the cell suspension
to a tissue culture plate or flask.
2
6eBioscience™ Immune Response T Cell Cytokines Kit User Guide
4. Incubate at 37°C, 5% CO2 for 16–24 hours.
5. Add Protein Transport Inhibitor Cocktail at 1X final concentration (2 μL per 1 mL) for the last 8–16
hours of culture.
Note: Do not exceed 16 hours with Protein Transport Inhibitor Cocktail as this may cause
excessive cell death.
Stain cells with viability dye
1. Prepare a stock solution of LIVE/DEAD™ Fixable Near IR (780):
a. Thaw DMSO and one vial of lyophilized dye.
b. Add 50 µL of DMSO to one vial of dye, then mix well.
2. Prepare a 2X working solution of LIVE/DEAD™ Fixable Near IR (780) by adding 2 µL of the stock
solution per 1 mL of PBS (azide-free). You will need the same volume as the cell suspension
prepared in step 1. Protect from light until ready to use.
3. Remove cells from the incubator, add 2X LIVE/DEAD™ Fixable Near IR (780) at 1:1, then mix well.
4. Incubate for 10–30 minutes at room temperature.
5. Transfer ~1 x 106 cells from the tissue culture well or flask into the 12 × 75 mm round bottom test
tubes, then centrifuge.
6. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
tube.
7. Add 2 mL of Flow Cytometry Staining Buer, then centrifuge.
8. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
tube.
Fix and permeabilize cells
1. Add 100 µL of IC Fixation Buer to each cell suspension, then incubate for 20–60 minutes at room
temperature, or up to 3 days at 4°C.
2. Prepare a 1X working solution of Permeabilization Buer—Dilute eBioscience™ Permeabilization
Buer (10X) 1:10 with distilled water. You will need ~6 mL of this working solution per sample.
Note: Precipitates may form in the eBioscience™ Permeabilization Buer (10X), but they will not
aect results. If precipitation occurs, filter the 1X Permeabilization Buer before use.
3. Add 2 mL of 1X Permeabilization Buer, then centrifuge.
4. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
tube.
Chapter 2 Methods
Test tube protocol 2
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 7
5. Add 2 mL of 1X Permeabilization Buer, then centrifuge.
6. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
tube.
7. Place a small aliquot of one sample into a separate 12 × 75 mm round bottom test tube. Set
this tube aside and protect from light. This will be the single-color compensation control for the
LIVE/DEAD™ Fixable Near IR (780).
Stain cells and compensation beads with fluorophore-conjugated antibodies
1. Prepare a cocktail of the antibodies for the isotype control sample(s):
Antibody Volume per sample
CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 μL
CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710, eBioscience™5 μL
CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 μL
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), FITC, eBioscience™1 μL
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE, eBioscience™1.25 μL
1X Permeabilization Buer 7.75 μL
Total antibody cocktail 25 μL
2. Prepare a cocktail of the antibodies for the fully-stained sample(s):
Antibody Volume per sample
CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 μL
CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710, eBioscience™5 μL
CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 μL
IFN gamma Monoclonal Antibody (4S.B3), FITC, eBioscience™1 μL
IL-17A Monoclonal Antibody (eBio64DEC17), PE, eBioscience™5 μL
1X Permeabilization Buer 4 μL
Total antibody cocktail 25 μL
3. Add 25 µL of the appropriate antibody cocktail to the desired tubes containing cells, then mix well.
4. Incubate for 30–60 minutes at room temperature. Protect from light.
5. Prepare single-color compensation controls.
a. Mix UltraComp eBeads™ Plus Compensation Beads by vigorously inverting at least 10 times
or pulse vortexing, then aliquot one drop into each of five 12 × 75 mm round bottom test
tubes labeled 1-5.
Chapter 2 Methods
Test tube protocol
2
8eBioscience™ Immune Response T Cell Cytokines Kit User Guide
b. Add the appropriate antibody to each tube, according to the following table.
Tube no. Antibody Volume to add
1 IFN gamma Monoclonal Antibody (4S.B3), FITC, eBioscience™1 µL
2 IL-17A Monoclonal Antibody (eBio64DEC17), PE, eBioscience™5 µL
3 CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710,
eBioscience™
5 µL
4 CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 µL
5 CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 µL
c. Mix well, then incubate for 15–30 minutes at room temperature. Protect from light.
6. Add 2 mL of 1X Permeabilization Buer to all tubes (cells and beads), then centrifuge.
7. Discard the supernatant, then resuspend the cells and beads in the residual volume of buer
remaining in the tube.
8. Add 2 mL of Flow Cytometry Staining Buer to all tubes (cells and beads), then centrifuge.
9. Discard the supernatant, then resuspend the cells and beads in 0.2–0.5 mL of Flow Cytometry
Staining Buer. Store at 4°C and protect from light until ready to analyze on a flow cytometer.
Note: For storage of up to 3 days before analysis, resuspend the cells in 100 µL of IC Fixation
Buer. Cells can be washed and resuspended in Flow Cytometry Staining Buer before analysis.
Samples can be stored at 4°C, protected from light until ready to analyze on a flow cytometer.
10. Collect data on a flow cytometer.
Chapter 2 Methods
Test tube protocol 2
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 9
Troubleshooting
Observation Possible cause Recommended action
Poor cell viability before
stimulation
Cell viability conditions were
suboptimal.
Ensure cells are prepared using a validated
protocol.
Resuspend cells in fresh, complete media.
On the day of the experiment, keep cells at
4°C, or on ice until ready to culture.
Cryopreserved cells were used
immediately after thawing.
Allow cells to rest in complete media at 37°C
for 20–30 minutes.
Unable to identify CD3, CD4,
or CD8 expressing cells
The incorrect compensation
was set.
Use the same antibody for compensation as in
the experiment.
Use FMO controls to validate the single-color
compensation.
The incorrect Forward/Side
scatter settings or gating were
set.
Use an ungated plot of CD3, CD4, or CD8.
Backgate onto a forward scatter versus side
scatter plot to determine where cells are, then
adjust voltages and gates accordingly.
Unable to detect IFN gamma or
IL-17A
The incorrect compensation
was set.
Use the same antibody for compensation as in
the experiment.
Use FMO controls to validate the single-color
compensation.
The protein transport inhibitor
was not added.
Add protein transport inhibitor for up to the
last 16 hours of incubation.
The stimulation conditions
were not optimized.
Optimize the duration of stimulation.
Titrate the concentration of stimulation
reagent(s).
If stimulation reagents are in DMSO, do not
exceed a final concentration of DMSO of 0.5%
(v/v).
Do not add the protein transport inhibitor for
longer than 16 hours.
A
10 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
96–well plate protocol
Stimulate cells
1. Resuspend cells at 2 x 107 cells/mL in complete media.
2. Add 50 μL of cells (~1 x 106) per well of a 96–well plate.
3. Prepare a 2X working solution of PHA-L by adding 10–20 μL of PHA-L (500X) per 1 mL of complete
media. You will need the same volume as the cell suspension prepared in step 1.
Alternatively, prepare a 2X working solution of peptide(s) or other stimulation reagent of choice.
Note: This dilution uses PHA-L at a final concentration of 5–10X. Using lower concentrations of
PHA-L may be desired and should be determined empirically.
4. Add 50 µL of the 2X PHA-L (or other stimulation reagents) to appropriate wells, then mix well.
5. Incubate at 37°C, 5% CO2 for 16–24 hours.
6. Add Protein Transport Inhibitor Cocktail at 1X final concentration (2 μL per mL) for the last 8–16
hours of culture.
Note: Do not exceed 16 hours with Protein Transport Inhibitor Cocktail as this may cause
excessive cell death.
Stain cells with viability dye
1. Prepare a stock solution of LIVE/DEAD™ Fixable Near IR (780):
a. Thaw DMSO and one vial of lyophilized dye.
b. Add 50 µL of DMSO to one vial of dye, then mix well.
2. Prepare a 2X working solution of LIVE/DEAD™ Fixable Near IR (780) by adding 2 µL of the stock
solution per 1 mL of PBS (azide-free). You will need the same volume as the cell suspension
prepared in step 1. Protect from light until ready to use.
3. Remove cells from the incubator, add 100 µL of 2X LIVE/DEAD™ Fixable Near IR (780), then mix
well.
4. Incubate for 10–30 minutes at room temperature, then centrifuge.
5. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
wells.
B
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 11
6. Add 250 µL of Flow Cytometry Staining Buer, then centrifuge.
7. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
wells.
Fix and permeabilize cells
1. Add 100 µL of IC Fixation Buer to each cell suspension.
2. Incubate for 20–60 minutes at room temperature, or up to 3 days at 4°C, then centrifuge.
3. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
wells.
4. Prepare a 1X working solution of Permeabilization Buer—Dilute eBioscience™ Permeabilization
Buer (10X) 1:10 with distilled water. You will need ~1 mL of this working solution per well.
Note: Precipitates may form in the eBioscience™ Permeabilization Buer (10X), but they will not
aect results. If precipitation occurs, filter the 1X Permeabilization Buer before use.
5. Add 250 µL of 1X Permeabilization Buer, then centrifuge.
6. Discard the supernatant, then resuspend the cells in the residual volume of buer remaining in the
wells.
7. Add 250 µL of 1X Permeabilization Buer, then centrifuge.
8. Discard the supernatant, then resuspend the cells in 75 µL of 1X Permeabilization Buer.
9. Set aside one well as the single-color compensation control for the LIVE/DEAD™ Fixable Near IR
(780) before proceeding with antibody staining.
Stain cells and compensation beads with fluorophore-
conjugated antibodies
1. Prepare a cocktail of the antibodies for the isotype control sample(s):
Antibody Volume per sample
CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 μL
CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710, eBioscience™5 μL
CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 μL
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), FITC, eBioscience™1 μL
Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE, eBioscience™1.25 μL
Appendix B 96–well plate protocol
Fix and permeabilize cells
B
12 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
(continued)
Antibody Volume per sample
1X Permeabilization Buer 7.75 μL
Total antibody cocktail 25 μL
2. Prepare a cocktail of the antibodies for the fully-stained sample(s):
Antibody Volume per sample
CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 μL
CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710, eBioscience™5 μL
CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 μL
IFN gamma Monoclonal Antibody (4S.B3), FITC, eBioscience™1 μL
IL-17A Monoclonal Antibody (eBio64DEC17), PE, eBioscience™5 μL
1X Permeabilization Buer 4 μL
Total antibody cocktail 25 μL
3. Add 25 µL of the appropriate antibody cocktail to the desired tubes containing cells, then mix well.
4. Incubate for 30-60 minutes at room temperature. Protect from light.
5. Prepare single-color compensation controls.
a. Mix UltraComp eBeads™ Plus Compensation Beads by vigorously inverting at least 10 times
or pulse vortexing, then aliquot one drop into each of five wells.
Note: These wells should not be placed near wells containing cells. If this is not possible, the
beads may be stained in a separate 96–well plate or in 12 × 75 mm round bottom test tubes.
b. Add the appropriate antibody to each of the five wells containing UltraComp eBeads™ Plus
Compensation Beads, according to the following table.
Well no. Antibody Volume to add
1 IFN gamma Monoclonal Antibody (4S.B3), FITC, eBioscience™1 µL
2 IL-17A Monoclonal Antibody (eBio64DEC17), PE, eBioscience™5 µL
3 CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710,
eBioscience™
5 µL
4 CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 µL
5 CD8a Monoclonal Antibody (SK1), eFluor 450, eBioscience™5 µL
c. Mix well, then incubate for 15–30 minutes at room temperature. Protect from light.
6. Add 150 µL of 1X Permeabilization Buer to all wells (cells and beads), then centrifuge.
7. Discard the supernatant, then resuspend the cells and beads in the residual volume of buer
remaining in the wells.
Appendix B 96–well plate protocol
Stain cells and compensation beads with fluorophore-conjugated antibodies B
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 13
8. Add 250 µL of Flow Cytometry Staining Buer to all wells (cells and beads), then centrifuge.
9. Discard the supernatant, then resuspend the cells and beads in 200–250 μL of Flow Cytometry
Staining Buer. Store at 4°C and protect from light until ready to analyze on a flow cytometer.
Note: For storage of up to 3 days before analysis, we recommend resuspending the cells in
100 µL of IC Fixation Buer. Cells can be washed and resuspended in Flow Cytometry Staining
Buer before analysis. Samples may be stored at 4°C, protected from light until ready to analyze
on a flow cytometer.
10. Collect data on a flow cytometer.
Appendix B 96–well plate protocol
Stain cells and compensation beads with fluorophore-conjugated antibodies
B
14 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
Typical experimental set up and
results
Typical experimental set up
Tube No. Description Sample type Antibodies or reagents
1 Unstained cells Cells none
2 FITC single-color
compensation control
Beads IFN gamma Monoclonal Antibody (4S.B3), FITC
3 PE single-color
compensation control
Beads IL-17A Monoclonal Antibody (eBio64DEC17),
PE
4 PerCP-eFluor
710 single-color
compensation control
Beads CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-
eFluor 710
5 APC single-color
compensation control
Beads CD3 Monoclonal Antibody (SK7), APC
6 eFluor 450 single-color
compensation control
Beads CD8a Monoclonal Antibody (SK1), eFluor 450
7 LIVE/DEAD Near IR
single-color control
Cells LIVE/DEAD Fixable Near-IR (780)
8 FITC FMO sample Cells •IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
C
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 15
(continued)
Tube No. Description Sample type Antibodies or reagents
9 PE FMO sample Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
10 PerCP-eFluor 710 FMO
sample
Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
11 APC FMO sample Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
12 eFluor 450 FMO sample Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•LIVE/DEAD Fixable Near-IR (780)
Appendix C Typical experimental set up and results
Typical experimental set up
C
16 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
(continued)
Tube No. Description Sample type Antibodies or reagents
13 LIVE/DEAD Near IR FMO
sample
Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
14 Unstimulated, isotype
control sample[1]
Cells •Mouse IgG1 kappa Isotype Control
(P3.6.2.8.1), FITC
•Mouse IgG1 kappa Isotype Control
(P3.6.2.8.1), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
15 Unstimulated, fully-
stained sample[1]
Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
16 Stimulated, isotype
control sample[1]
Cells •Mouse IgG1 kappa Isotype Control
(P3.6.2.8.1), FITC
•Mouse IgG1 kappa Isotype Control
(P3.6.2.8.1), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
Appendix C Typical experimental set up and results
Typical experimental set up C
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 17
(continued)
Tube No. Description Sample type Antibodies or reagents
17 Stimulated, fully-stained
sample[1]
Cells •IFN gamma Monoclonal Antibody (4S.B3),
FITC
•IL-17A Monoclonal Antibody
(eBio64DEC17), PE
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
[1] Duplicate this sample as needed for experimental design.
Appendix C Typical experimental set up and results
Typical experimental set up
C
18 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
Typical experimental results
The following results were generated using normal, healthy PBMC prepared as described in this user
guide. The data were collected on a spectral flow cytometer, and single-color compensation controls
were used for unmixing.
1
4
2
3
Figure 1 Gating strategy
1Gate out debris and electronic noise
2Gate on single cells
3Gate on live CD3+ cells
4Gate on CD8+ or CD4+ cells
Appendix C Typical experimental set up and results
Typical experimental results C
eBioscience™ Immune Response T Cell Cytokines Kit User Guide 19
12
34
Figure 2 Analysis of IFN-γ and IL-17A expression
1Unstimulated control (DMSO) gated on live CD3+ CD8+ cells
2Stimulated (PHA-L) gated on live CD3+ CD8+ cells
3Unstimulated control (DMSO) gated on live, CD3+ CD4+ cells
4Stimulated (PHA-L) gated on live, CD3+ CD4+ cells
Appendix C Typical experimental set up and results
Typical experimental results
C
20 eBioscience™ Immune Response T Cell Cytokines Kit User Guide
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
  • Page 1 1
  • Page 2 2
  • Page 3 3
  • Page 4 4
  • Page 5 5
  • Page 6 6
  • Page 7 7
  • Page 8 8
  • Page 9 9
  • Page 10 10
  • Page 11 11
  • Page 12 12
  • Page 13 13
  • Page 14 14
  • Page 15 15
  • Page 16 16
  • Page 17 17
  • Page 18 18
  • Page 19 19
  • Page 20 20
  • Page 21 21
  • Page 22 22
  • Page 23 23
  • Page 24 24

Thermo Fisher Scientific eBioscience Immune Response T Cell Cytokines Kit User guide

Brand
Thermo Fisher Scientific
Model
eBioscience Immune Response T Cell Cytokines Kit
Type
User guide
Download PDF
report

Ask a question and I''ll find the answer in the document

Finding information in a document is now easier with AI

Related papers

Other documents