Propidium iodide staining.

Method

1. Harvest cells in the appropriate manner and wash in PBS.
2. Fix in cold 70 % ethanol. Add dropwise to the cell pellet while vortexing. This should ensure fixation of all cells and minimise clumping.
3. Fix for at least 30 minutes at 4 °C. Specimens can be left at this stage for several weeks.
4. Wash 2x in PBS. Spin at 2000 rpm and be careful to avoid cell loss when discarding supernatant, especially after spinning out of ethanol.
5. To ensure that only DNA is stained, treat cells with ribonuclease. Add 50 µl of 100 µg/ml RNase.
6. Add 200 µl propidium iodide (50 µg/ml).
7. Analyse by flow cytometry.