Thermo Fisher Scientific eBioscience Immune Response T Cell Receptor Signaling Kit User guide

Brand
Thermo Fisher Scientific
Model
eBioscience Immune Response T Cell Receptor Signaling Kit
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
eBioscience™ Immune Response T Cell
Receptor Signaling Kit
USER GUIDE
Catalog Number A53422
Document Part Number 2162762
Publication Number MAN0026419
Revision A.0
Pierce Biotechnology, Inc. | Thermo Fisher Scientific | 3747 N. Meridian Road | Rockford, Illinois 61101 USA
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The information in this guide is subject to change without notice.
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ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0026419
Revision Date Description
A.0 8 March 2022 New document created for eBioscience™ Immune Response T Cell Receptor Signaling Kit.
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©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 with CD3 and CD28 ........................................... 7
Stain cells with viability dye .................................................. 7
Fix and permeabilize cells .................................................... 8
Stain cells and compensation beads with fluorophore-conjugated antibodies ....... 8
■APPENDIX A Troubleshooting .................................................... 10
■APPENDIX B 96–well plate protocol ............................................. 11
Stimulate cells with CD3 and CD28 ............................................... 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 ..................................................... 18
■APPENDIX D Safety ............................................................... 20
Chemical safety ................................................................ 20
Biological hazard safety ......................................................... 21
■APPENDIX E Documentation and support ...................................... 22
Customer and technical support ................................................. 22
Limited product warranty ........................................................ 22
eBioscience™ Immune Response T Cell Receptor Signaling 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 Receptor Signaling Kit includes all of the components
needed to stimulate and detect phosphorylation of CD247 (CD3 zeta) on tyrosine 142 (Tyr142) in 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 Receptor Signaling Kit (Cat. No. A53422)
Contents Cat. No. Amount Storage
LIVE/DEAD™ Fixable Near IR (780) Viability kit,
for 633 nm excitation
L34993 200 assays −20°C
CD3 Monoclonal Antibody (OKT3), Functional
Grade, eBioscience™
16-0037-85 500 μg 4°C
CD28 Monoclonal Antibody (CD28.6),
Functional Grade, eBioscience™
16-0288-85 500 μg
F(ab')2-Goat anti-Mouse IgG (H+L) Secondary
Antibody, Functional Grade, eBioscience™
16-5098-85 500 μg
eBioscience™ Flow Cytometry Staining Buer 00-4222-57 200 mL
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
1
4eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
Table 1 eBioscience Immune Response T Cell Receptor Signaling Kit (Cat. No. A53422) (continued)
Contents Cat. No. Amount Storage
CD8a Monoclonal Antibody (SK1), eFluor 450,
eBioscience™
4°C48-0087-42 100 tests
Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa Fluor
488, eBioscience™
53-2478-42 100 tests
Mouse IgG2b kappa Isotype Control (eBMG2b),
Alexa Fluor 488, eBioscience™
53-4732-80 25 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 Receptor Signaling 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 Phospho-CD247 (CD3 zeta) (Tyr142))
– 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.
2
6eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
Stimulate cells with CD3 and CD28
Note: If your experiment requires stimulation with a peptide(s) or other stimulation reagent(s), the
conditions should be determined empirically.
1. Resuspend cells at 2 x 107 cells/mL in complete media.
2. Dilute cells to 1 x 107 cells/mL in complete media.
3. Add 10 µg/mL each of CD3 Monoclonal Antibody (OKT3), Functional Grade, eBioscience™ and
CD28 Monoclonal Antibody (CD28.6), Functional Grade, eBioscience™ to the cell suspension. Mix
well, then incubate on ice for 15 minutes.
4. Add 10 µg/mL of F(ab')2-Goat anti-Mouse IgG (H+L) Secondary Antibody, Functional Grade,
eBioscience™. Mix well, then incubate on ice for 10 minutes.
5. Transfer the cell suspension to a tissue culture plate or flask.
6. Incubate at 37°C, 5% CO2 for 5 minutes.
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.
Chapter 2 Methods
Test tube protocol 2
eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide 7
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.
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 IgG2b kappa Isotype Control (eBMG2b), Alexa Fluor 488,
eBioscience™
1 μL
1X Permeabilization Buer 9 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
Mouse IgG2b kappa Isotype Control (eBMG2b), Alexa Fluor 488,
eBioscience™
5 μL
Chapter 2 Methods
Test tube protocol
2
8eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
(continued)
Antibody Volume per sample
1X Permeabilization Buer 5 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 four 12 × 75 mm round bottom test
tubes labeled 1-4.
b. Add the appropriate antibody to each tube, according to the following table.
Tube no. Antibody Volume to add
1 Phospho-CD247 (CD3 zeta) (Tyr142) Monoclonal Antibody
(3ZBR4S), Alexa Fluor 488, eBioscience™
5 µL
2 CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710,
eBioscience™
5 µL
3 CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 µL
4 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 the 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 the 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 Receptor Signaling 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
phosphorylation of CD247
(CD3 zeta) on tyrosine 142
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 stimulation conditions
were not optimized.
Optimize 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).
A
10 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
96–well plate protocol
Stimulate cells with CD3 and CD28
Note: If your experiment requires stimulation with a peptide(s) or other stimulation reagent(s), the
conditions should be determined empirically.
1. Resuspend cells at 2 x 107 cells/mL in complete media.
2. Dilute cells to 1 x 107 cells/mL in complete media.
3. Add 10 µg/mL each of CD3 Monoclonal Antibody (OKT3), Functional Grade, eBioscience™ and
CD28 Monoclonal Antibody (CD28.6), Functional Grade, eBioscience™ to the cell suspension. Mix
well, then incubate on ice for 15 minutes.
4. Add 10 µg/mL of F(ab')2-Goat anti-Mouse IgG (H+L) Secondary Antibody, Functional Grade,
eBioscience™. Mix well, then incubate on ice for 10 minutes.
5. Transfer 100 µL of the cell suspension per well.
6. Incubate at 37°C, 5% CO2 for 5 minutes.
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 Receptor Signaling 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 IgG2b kappa Isotype Control (eBMG2b), Alexa Fluor 488,
eBioscience™
1 μL
1X Permeabilization Buer 9 μL
Total antibody cocktail 25 μL
Appendix B 96–well plate protocol
Fix and permeabilize cells
B
12 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
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
Phospho-CD247 (CD3 zeta) (Tyr142) Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488, eBioscience™
5 μL
1X Permeabilization Buer 5 μL
Total antibody cocktail 25 μL
3. Add 25 µL of appropriate the 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 four 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 well, according to the following table.
Well no. Antibody Volume to add
1 Phospho-CD247 (CD3 zeta) (Tyr142) Monoclonal Antibody
(3ZBR4S), Alexa Fluor 488, eBioscience™
5 µL
2 CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-eFluor 710,
eBioscience™
5 µL
3 CD3 Monoclonal Antibody (SK7), APC, eBioscience™5 µL
4 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.
8. Add 250 µL of Flow Cytometry Staining Buer to all wells (cells and beads), then centrifuge.
Appendix B 96–well plate protocol
Stain cells and compensation beads with fluorophore-conjugated antibodies B
eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide 13
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, 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.
Appendix B 96–well plate protocol
Stain cells and compensation beads with fluorophore-conjugated antibodies
B
14 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
Typical experimental set up and
results
Typical experimental set up
Tube No. Description Sample type Antibodies or reagents to add
1 Unstained cells Cells none
2 Alexa Fluor 488 single-
color compensation
control
Beads Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa Fluor 488
3 PerCP-eFluor
710 single-color
compensation control
Beads CD4 Monoclonal Antibody (SK3 (SK-3)), PerCP-
eFluor 710
4 APC single-color
compensation control
Beads CD3 Monoclonal Antibody (SK7), APC
5 eFluor 450 single-color
compensation control
Beads CD8a Monoclonal Antibody (SK1), eFluor 450
6 LIVE/DEAD Near IR
single-color control
Cells LIVE/DEAD Fixable Near-IR (780)
7 Alexa Fluor 488 FMO
sample
Cells •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)
8 PerCP-eFluor 710 FMO
sample
Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
C
eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide 15
(continued)
Tube No. Description Sample type Antibodies or reagents to add
9 APC FMO sample Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD8a Monoclonal Antibody (SK1), eFluor
450
•LIVE/DEAD Fixable Near-IR (780)
10 eFluor 450 FMO sample Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•LIVE/DEAD Fixable Near-IR (780)
11 LIVE/DEAD Near IR FMO
sample
Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•CD4 Monoclonal Antibody (SK3 (SK-3)),
PerCP-eFluor 710
•CD3 Monoclonal Antibody (SK7), APC
•CD8a Monoclonal Antibody (SK1), eFluor
450
12 Unstimulated, isotype
control sample[1]
Cells •Mouse IgG2b kappa Isotype Control
(eBMG2b), Alexa Fluor 488
•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)
13 Unstimulated, fully-
stained sample[1]
Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•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
16 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
(continued)
Tube No. Description Sample type Antibodies or reagents to add
14 Stimulated, isotype
control sample[1]
Cells •Mouse IgG2b kappa Isotype Control
(eBMG2b), Alexa Fluor 488
•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 Stimulated, fully-stained
sample[1]
Cells •Phospho-CD247 (CD3 zeta) (Tyr142)
Monoclonal Antibody (3ZBR4S), Alexa
Fluor 488
•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
eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide 17
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.
12
34
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
18 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
phospho-CD247 (Tyr142)phospho-CD247 (Tyr142)
12
Figure 2 Analysis of phospho-CD247 (CD3 zeta) (Tyr142) expression
1Gated on live CD3+ CD8+ cells
2Gated on live, CD3+ CD4+ cells
Appendix C Typical experimental set up and results
Typical experimental results C
eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide 19
Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user
documentation may result in personal injury or damage to the instrument or device. Ensure that
anyone using this product has received instructions in general safety practices for laboratories and
the safety information provided in this document.
·Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
·Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, visit thermofisher.com/support.
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel
read and practice the general safety guidelines for chemical usage, storage, and waste provided
below. Consult the relevant SDS for specific precautions and instructions:
·Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see
the "Documentation and Support" section in this document.
·Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
·Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sucient ventilation (for example, fume hood).
·Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
·Handle chemical wastes in a fume hood.
·Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container.
Both containers must be compatible with the waste material and meet federal, state, and local
requirements for container storage.)
·After emptying a waste container, seal it with the cap provided.
·Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
·Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
·IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
D
20 eBioscience™ Immune Response T Cell Receptor Signaling Kit User Guide
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Thermo Fisher Scientific eBioscience Immune Response T Cell Receptor Signaling Kit User guide

Brand
Thermo Fisher Scientific
Model
eBioscience Immune Response T Cell Receptor Signaling Kit
Type
User guide
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