Before you start
- Make fresh stimulation mix in warm cRPMI.
- Include an unstimulated control (inhibitor only) for each condition.
Reagents and materials
Totals are for 12 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| cRPMI for restimulation | 0.2 mL | 0.2 mL |
| PMA (final 50 ng/mL) | 10 ng | 10 ng |
| Ionomycin (final 500 ng/mL – 1 µg/mL) | 100 ng | 100 ng |
| Brefeldin A (final ~5 µg/mL) and/or monensin (~2 µM)as BFA | 1 µg | 1 µg |
| Fixation/permeabilisation solution | 200 µL | 200 µL |
| 1× permeabilisation/wash buffer | 0.6 mL | 0.6 mL |
| Intracellular antibody cocktail in perm buffer | 50 µL | 50 µL |
Buffers and recipes
Complete RPMI (cRPMI) per 500 mL
| RPMI 1640 | 440 mL |
| Heat-inactivated FBS (10%) | 50 mL |
| L-glutamine, 200 mM (2 mM) | 5 mL |
| Penicillin–streptomycin, 100× (1×) | 5 mL |
| 2-Mercaptoethanol, 55 mM (50 µM) | 0.45 mL |
Optional: 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids. Use within ~4 weeks.
FACS buffer per 500 mL
| PBS (Ca²⁺/Mg²⁺-free) | 490 mL |
| Heat-inactivated FBS (2%) | 10 mL |
| 0.5 M EDTA, pH 8.0 (1 mM final) | 1 mL |
Filter (0.22 µm) and keep at 4 °C. 0.5% BSA can replace FBS. Sodium azide (0.05–0.1%) is optional for staining only — never for cells you will culture.
Equipment
- 37 °C, 5% CO₂ incubator
- 96-well round-bottom plate
- Plate centrifuge
- Flow cytometer
Procedure
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Set up stimulation
Plate 1–2 × 10⁶ cells per well in 200 µL cRPMI containing PMA, ionomycin and brefeldin A and/or monensin.
Incubate
Culture 4–5 h at 37 °C, 5% CO₂. Add anti-CD107a during this step if you measure degranulation.
Viability dye and surface stain
Wash, then stain with fixable viability dye and surface antibodies as in the surface-staining protocol. Stain CD4 well — PMA lowers its surface level.
Fix and permeabilise
Resuspend in 200 µL fixation/permeabilisation solution for 20 min at 4 °C in the dark.
Wash in perm buffer
Wash twice with 200 µL 1× perm/wash buffer (keep the detergent present from now on).
Intracellular stain
Resuspend in 50 µL antibody cocktail made in perm buffer and incubate 30 min at 4 °C (or room temperature) in the dark.
Wash and acquire
Wash twice with perm buffer, resuspend in FACS buffer and acquire.
Critical points
- Brefeldin A blocks most cytokines efficiently; monensin is better for some (e.g. CD107a assays). Some labs combine both.
- Longer than ~6 h restimulation reduces viability.
- Gate cytokine positives on the unstimulated control, not on isotype controls.
Troubleshooting
| Problem | What to check or change |
|---|---|
| No cytokine signal | Check PMA/ionomycin activity (fresh aliquots), that the transport inhibitor was added, and that permeabilisation buffer was used for staining. |
| High background in unstimulated | Reduce antibody amount; wash with perm buffer; use FMO controls. |
| Lost CD4 staining | Stain CD4 on fixed cells, or use a clone known to work after fixation. |
Safety
PMA is a tumour promoter and ionomycin is toxic: wear gloves, avoid skin contact and aerosols.
Further reading
- Foster B et al. Detection of intracellular cytokines by flow cytometry. Current Protocols in Immunology.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.