The following protocol is for intracellular cytokine staining (formaldehyde/saponin method).

This protocol is used courtesy of Peter Openshaw, Respiratory Medicine, St. Mary's Hospital Medical School, Imperial College, Norfolk Place, London, UK.

Reagents

  • Anti-cytokine monoclonal antibodies
  • Isotype control antibody
  • PMA/ iononmycin or specific antigen/ APC
  • Brefeldin A
  • 2% formaldehyde 
  • Permeabilization buffer:
    • PBS 1%
    • BSA
    • 0.5% saponin
    • azide

Most resting cells are negative for IFN gamma, IL-2, IL-4, IL-5 and IL-10 even if they have seen antigen and are committed to one pattern or another of cytokine production. In this protocol, we therefore restimulate with PMA/iononmycin to induce some synchrony, although different cytokines behave very differently in terms of timing and duration of display.

Staining

  1. Isolate cells
  2. Stimulate with PMA/iononmycin or specific antigen/APC (when compared, both do the same)
  3. At various times after stimulation (2-22 hrs.) add brefeldin A for the last 2 hours of culture (to destroy the Golgi and allow intracellular accumulation)
  4. Fix with 2% formaldehyde for 20 minutes at room temperature
    Store for up to 2 days or stain straight away. All following steps are at room temperature and in permeabilization buffer.
  5. Treat with permeabilization buffer for 10 minutes.
  6. Add unconjugated anti-cytokine monoclonal antibody or isotype control.
  7. Incubate for 30 minutes at room temperature.
  8. Wash with permeabilization buffer.
  9. Add FITC anti-mouse or similar.
  10. Incubate for 30 minutes at room temperature.
  11. Block with mouse or rat Ig 0.3 mg/ml.
  12. Add PE conjugated anti-IFN gamma (AN18 PE).
  13. Incubate for 30 minutes at room temperature.
  14. Wash twice with permeabilization buffer.
  15. Wash out saponin.
  16. Some surface antigens are stable through this protocol and can be used as 3rd/4th color(s).

References

This is a modified method based on that published by Radbruch's group (Assenmacher et al. Eu. J. Immunol. 24:1097, 1994)