Author
Date
Version
SOP #
Stephanie Swift
16th July 2015
3
H003
Objective: This SOP addresses the peptide stimulation and intracellular cytokine
staining of human PBMCs for flow cytometric analysis
SOP-H002: Human Intracellular Cytokine Staining
Required Reageants and Equipment
Ficoll/Leucosop
Trypan Blue
FACS Buffer (2.5g BSA in 500ml PBS)
cRPMI (10% FCS, 5ml L-Glut, 5ml HEPES, 5ml Pen/Strep in 500ml RPMI)
PBS
PMA (Sigma #P1585) + Ionomycin (Sigma #I0634)
Cytofix/Cytoperm + Permwash (BD # 554714)
1 x Permwash buffer
Golgi Plug (BD #554724)
CD8-PerCP-Cy5.5 (BD Clone RPA-T8 #560662)
IFNg-APC (BD Clone B27 #562017)
TNFa-PE (BD Clone Mab11 #557068)
Viability Dye-eF450 (eBiosciences # 65-0863-14)
DMSO
0.5M EDTA
Note: The dilutions of each antibody may change between different lots of the
same antibody clone received from the same company. Individual lot titrations
should be carried out to optimise dilutions.
DAY ONE:
1. Plate 100ul per well (2x10^6 cells) in a 96-well U-bottom plate
2. Prepare peptide master mixes, and add 50ul/well. Incubate at 37°C for 1h.
For the negative control, add DMSO (diluted in cRPMI).
For the positive control, add 50ul/well of diluted PMA/ionomycin
For the test wells, add 50ul/well of diluted peptide of interest. See appendix A for
dilution calculations.
4. Add 50ul/well of Golgi Plug master mix. See appendix B for dilution calculations.
5. Continue incubation for a further 4h at 37°C
6. Stop stimulation by adding 50ul/well cold EDTA/cRPMI for 15 mins. at RT. See
appendix C for dilution calculations
7. Spin down plate at 1500RPM for 5 mins. and wash samples in 200ul cRPMI.
Count cells using 1:1 mix with Trypan Blue (20ul:20ul) and record count per well
(if required).
You can either stop here and leave plates at 4ºC overnight (in the dark), or
continue straight on to the next step.
DAY TWO:
This protocol is shared by the Stojdl Lab under a creative commons license.
8. Spin down plate and aspirate off supernatant. Add 50ul of CD8 cocktail together
with live/dead viability dye, and incubate on ice for 30 mins. IN THE DARK.
Example calculation for 20 wells:
Reageant
Dilution
Volume
CD8
0.5ul/well
10.0ul
Viability Dye-eF450
0.1ul/well
2.0ul
FACS Buffer
988.0ul
9. Wash 150ul FACS buffer. Spin down and aspirate off s/n.
10. Wash 200ul FACS buffer. Spin down and aspirate off s/n.
11. Add 100ul per well of Cytofix/Cytoperm for 20 mins. on ice IN THE DARK.
12. Wash 100ul Permwash buffer. Spin down and aspirate off s/n.
13. Wash 200ul Permwash buffer. Spin down and aspirate off s/n.
14. Add 50ul of IFNg/TNFa cocktail and incubate for 30 mins. on ice IN THE
DARK. Example calculation for 20 wells:
Reageant
IFNg
TNFa
FACS Buffer
Dilution
1.25ul/well
12.6ul/well
-
Volume (ul)
25
252
723
15. Wash 150ul Permwash buffer. Spin down and aspirate off s/n.
16. Wash 200ul Permwash buffer. Spin down and aspirate off s/n.
17. Resuspend in 200ul FACS buffer. Combine volume in wells if you’ve stained
them in duplicate/triplicate. Keep on ice and in the dark until it's time to run the
samples on the flow cytometer. Filter through nylon mesh just prior to running on
the flow cytometer.
Protocol originally modified from the Bramson lab, McMaster University, Hamilton,
Canada
This protocol is shared by the Stojdl Lab under a creative commons license.
Appendix
A. TEST PEPTIDES/CONTROLS FOR STIMULATIONS
Negative Control Wells
Dilution
DMSO
cRPMI
highest
concentration
used in test
peptide wells
-
Example Volume for 10 wells
(ul)
40.0
460.0
Positive Control Wells (PMA/Ionomycin)
1. Phorbol 12-myristate 13-acetate (PMA) powder (Sigma #P-1585)
a. Reconstitute in DMSO at 0.1 mg/mL.
b. Store small aliquots (eg, 20 μL) at –20°C; do not refreeze aliquots after thawing.
c. Dilute 1:100 in sterile PBS (without sodium azide) for each experiment to
prepare a fresh stock solution.
2. Ionomycin powder (Sigma #I-0634)
a. Reconstitute in EtOH at 0.5 mg/mL.
b. Store at –20°C.
c. Dilute 1:10 in sterile PBS (without sodium azide) for each experiment to prepare
a fresh stock solution.
3. Mix fresh PMA solution at a final concentration of 10 ng/mL of cell suspension
(0.5ul per 50ul - in cRPMI) with fresh ionomycin solution at a final concentration of 1
μg/mL of cell suspension (1ul per 50ul - in cRPMI).
Volume for 1 well
Example Volume for 10 wells
From Fresh
(ul)
(ul)
Stock
PMA
0.5
5
Ionomycin
1.0
10
cRPMI
48.5
485
Test peptides (calculations will vary after you optimise the required peptide
concentration per well).
Peptide MHC
HLA-A*02:01
Protein
CMV pp65495-503
CD8/CD
4
CD8+
Peptide
Sequence
NLVPMVATV
Stock
Final
Concentration
Concentration
B. GOLGI PLUG
Solution
Golgi Plug
cRPMI
Add 50ul/well
Dilution
0.4ul/well
-
Example Volume
for 30 wells
(ul)
12.0
1488.0
C. EDTA/cRPMI – Keep at 4°C after preparation
This protocol is shared by the Stojdl Lab under a creative commons license.
Solution
0.5M EDTA
cRPMI
Add 50ul/well
Dilution
1:25
-
Example Volume
for 50 wells
(ul)
100.0
2400.0
This protocol is shared by the Stojdl Lab under a creative commons license.