- Harvest cells, washing in PBS.
- Fix pelleted cells in ice-cold 70% ethanol by adding with a pasteur pipette on a vortex. Leave cells at 4oC from 30 min to a week.
- Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in PBS.
- Pellet cells, wash twice in PBS.
- Add 50 µl RNAse to pelleted cells (100 µg/ml Sigma) and incubate at RT or 37oC for 15 min.
- Add 200-400 µl of PI (final concentration 50 µg/ml Sigma P4170).
- Analyse by flow cytometry collecting 25,000 events per sample.