SubG₁ Protocol

1. Harvest cells washing in PBS.
2. Fix pelleted cells in ice-cold 70% ethanol by adding with a pasteur pipette on a vortex. Leave cells at 4^oC from 30 mins to a week.
3. Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in Phosphate Citrate PBS buffer (0.1M Citric Acid pH 7.8).
4. Add 50 ml RNAse to pelleted cells (100 mg/ml Sigma) and incubate at RT or 37^oC for 15 mins.
5. Add 200-400 ml of PI (final concentration 50 mg/ml Sigma P4170).
6. Analyse by flow cytometry collecting 25,000 events per sample.