intracellular cytokine staining

Preparation:

PMA -use at 10ng/ml final concentration. Stock at 1mg/ml in enthol or DMSO, aliquots

Ionomycin -use at 500ng/ml final concentration. Stock at 1mg/ml in enthol or DMSO,

Anti-CD3e -use at 5ug/ml final concentration. Stock from R&D systems or Pharmingen.

Anti-CD28 -use at 5ug/ml final concentration. Stock from R&D systems or Pharmingen.

Brefeldin A (BFA) -use at 10ug/ml final concentration. Dissolve BFA at 10mg/ml in ethanol, then the Stock from R&D systems or Pharmingen.

4%PFA in PBS Need to dissolve at 60degree, aliquot and store at –80degree.

PermWash PBS containing 0.1% saponin, 0.5%BSA, and 0.1%Azide.

FACs buffer PBS containing 0.5%BSA, and 0.1%Azide.

Procedures

  1. Surface staining and wash; Discard the FACS buffer,

For PMA+Ca++-stimulated T cells, CD4 staining can be done with the cytokine staining step since PMA downregulate CD4 from the surface.

  1. Put 75ul(for 1million cells) 4%PFA in PBS, vortex briefly, then 20-30 minutes at RT,

  2. Alternatively, add 1ml FACS buffer and continue staining in second day.

  3. Wash with 1ml PermWash,

  4. Repeat Step d.

  5. Put 50ul of PermWash with Fcblocker,

  6. RT, 10 min,

  7. Add 50ul PermWash with antibodies against cytokines.

  8. 4 degree for 20-30 minutes.

  9. Wash with 2ml PermWash,

  10. Discard the supernatant.

  11. Put FACS buffer and ready for FACS. (The cell pellet can kept for at least for 2days at 4 degree till ready for FACS)