Intracellular FACS staining with Paraformaldehyde and Saponin
Stimulation with PMA (final at 50ng/ml) and Ionomycine (final at 1ug/ml) for 4h at 37oC. After 2h add Brefeldin A (final at 10ug/ml).
Transfer into V bottom well plates.
Wash once in FACS buffer + Brefeldin A.
Resuspend in 100ul FACS buffer + Brefeldin A and add 100ul of Paraformaldehyde 4%. PAF4% tubes contain 2ml, calculate how many tubes needed.
Incubate 15min at 4oC.
Wash twice in FACS buffer + Brefeldin A and centrifuge 5min at 2000rpm from now on!
Wash once in FACS buffer + Saponin 0.5% (final).
Incubate 20 min at RT with FACS buffer + Saponin 0.5% (final) + Fc Block + 5% NMS.
Without washing add intracellular Ab (1/100) in FACS buffer + Saponin 0.5% (final).
Incubate 30 min at RT.
Wash once in FACS buffer + Saponin 0.5% (final).
Wash once in FACS buffer.
Cells are ready to go to the FACS.
Intracellular FACS staining with Paraformaldehyde and Saponin —> PDF