DAPI 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 1 mg of DAPI (Sigma D9542).
9. Analyse by flow cytometry collecting 25,000 events per sample.