PI and Surface Antigen Staining Protocol

1. Harvest cells washing in PBS.
2. Add fluorescently labelled mcab to pelleted cells for 20 mins at RT.
3. Wash with PBS.
4. Fix with 0.5% paraformaldehyde (PFA) for 15 min on ice.
5. Wash twice with PBS.
6. Fix pelleted cells in ice-cold 70% ethanol by adding with pasteur pipette on a vortex. Leave cells at 4^oC from 30 mins.
7. Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in PBS.
8. Add 50 ml RNAse to pleeted cells(100 mg/ml Sigma) and incubate at RT or 37^oC for 15 mins.
9. Add 200-400 ml of PI (final concentration 50 mg/ml Sigma P4170).
10. Analyse by flow cytometry collecting 25,000 events per sample.