PI and Intracellular Antigen Staining Protocol

1. Harvest cells washing in PBS.
2. Fix with 0.5% paraformaldehyde (PFA) for 15 min on ice.
3. Wash twice with PBS.
4. Fix pelleted cells in ice-cold 70% ethanol by adding with pasteur pipette on a vortex. Leave cells at 4^oC for 30 mins.
5. Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in PBS/0.25% saponin.
6. Add 1 mg mcab and incubate for 30 min on ice.
7. Wash twice in PBS/0.25% saponin.
8. Add 0.5 mg mcab and incubate for 30 min on ice.
9. Wash once in PBS/0.25% saponin.
10. Add 50 ml RNAse to pelleted cells (100 mg/ml Sigma) and incubate at RT or 37^oC for 15 mins.
11. Add 200-400 ml of PI (final concentration 50 mg/ml Sigma P4170).
12. Analyse by flow cytometry collecting 25,000 events per sample.