Propidium iodide staining of cells for cell cycle analysis

Reagents

Phosphate buffered saline (PBS) (BUF036A)

70% Ethanol in DI water

Nucleic acid staining solution (1x PBS, 100 ug/ml RNAse A)

Methods

1. Prepare cells appropriately; refer to protocol FC1. ‘Preparation of cells for flow cytometry’ for further information.
2. Fix in 2-5 ml cold (4^oC) 70% ethanol. Add drop wise to cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping.
3. Fix for at least 30 min on ice. Note: specimens can be left at this stage for several weeks.
4. Centrifuge at 500 g for 10 min, decant supernatant.
5. Wash twice with 3 ml PBS at 300-400 g and 4^oC for 5 min and discard supernatant.
6. Resuspend cell pellet in 500 ul nucleic acid staining solution. Mix well.
7. Incubate for 30 min at room temperature.
8. Add propidium iodide e.g. 1-2 drops of ReadiDrop™ propidium iodide (135-1101).
9. Analyze by flow cytometry. The propidium iodide should be read on the appropriate channel in the linear scale. Doublets should be gated out using the Area vs Height or Width depending on your instrument.

Notes

Appropriate controls should always be carried out, for flow cytometry the following should be considered for inclusion;

• Unstained cells should always be included in the experimental set-up to monitor autofluorescence.