Flow Cytometry Intracellular Cytokine Staining Protocol

Materials Needed

• Cell sample (PBMCs, splenocytes, or cultured cells)
• Cell stimulation cocktail (e.g., PMA/ionomycin or antigen-specific stimulation)
• Protein transport inhibitor (e.g., Brefeldin A and/or Monensin)
• Fixation buffer (e.g., 4% paraformaldehyde or commercial fixation solution)
• Permeabilization buffer (e.g., 0.1% saponin in PBS or commercial permeabilization solution)
• Fluorochrome-conjugated intracellular antibodies
• Surface staining antibodies (optional)
• Blocking buffer (PBS + 10% FBS)
• 96-well V-bottom plate or tubes
• Ice bucket
• Centrifuge
• Flow cytometer

Protocol

1. Cell Stimulation
   • Adjust cell concentration to 1-2×10⁶ cells/mL in complete culture medium.
   • Add stimulation cocktail (e.g., 50 ng/mL PMA + 1 μg/mL ionomycin) and protein transport inhibitor (e.g., 10 μg/mL Brefeldin A).
   • Incubate for 4-6 hours at 37°C in a CO₂ incubator.
   • Include an unstimulated control without stimulation cocktail.
2. Surface Staining (Optional)
   • Centrifuge cells at 300 g for 5 minutes and aspirate supernatant.
   • Resuspend in 100 μL blocking buffer and incubate for 10 minutes at room temperature.
   • Add surface marker antibodies at manufacturer-recommended concentrations.
   • Incubate for 30 minutes at 4°C in the dark.
   • Wash with 1-2 mL PBS, centrifuge at 300 g for 5 minutes, and aspirate supernatant.
3. Fixation
   • Resuspend cells in 100-200 μL fixation buffer (e.g., 4% paraformaldehyde in PBS).
   • Incubate for 15-20 minutes at room temperature in the dark.
   • Wash twice with PBS by centrifuging at 300 g for 5 minutes.
4. Permeabilization and Intracellular Staining
   • Resuspend cells in 100 μL permeabilization buffer containing blocking reagent.
   • Add intracellular cytokine antibodies at manufacturer-recommended concentrations.
   • Incubate for 30-60 minutes at 4°C or room temperature in the dark.
5. Washing
   • Add 1-2 mL permeabilization buffer and centrifuge at 300 g for 5 minutes.
   • Repeat wash once more with permeabilization buffer.
   • Final wash with PBS, then resuspend pellet in 300-500 μL PBS.
6. Acquisition
   • Analyze samples on a flow cytometer within 24 hours. Adjust voltage settings to ensure fluorescent peaks are on scale and clearly distinguishable.
7. Data Analysis
   • Use flow cytometry software to analyze data. Gate on live cells, then on cell populations of interest.
   • Compare stimulated versus unstimulated samples to identify cytokine-positive populations.

Important Considerations

Stimulation Conditions

• Optimize stimulation time (typically 4-6 hours) and reagent concentrations for your cell type.
• Always include unstimulated controls to assess background cytokine production.
• Protein transport inhibitors are essential to prevent cytokine secretion. Different protein transport inhibitors may work better per target and cell type, so optimization may be required.

Fixation and Permeabilization

• Use fresh fixation buffer; paraformaldehyde degrades over time.
• Maintain permeabilization buffer throughout intracellular staining steps.
• Some fixation/permeabilization kits are optimized for specific transcription factors.

Controls

• Include fluorescence-minus-one (FMO) controls for accurate gating.
• Use isotype controls or unstained samples to assess non-specific binding.
• Compensation controls are critical for multi-color panels.

Antibody Selection

• Validate antibody clones for intracellular staining applications.
• Brighter fluorochromes should be used for low-abundance cytokines.
• Consider using viability dyes before fixation and permeabilization to exclude dead cells.