The following protocol is for whole cell DNA staining with propidium iodide for cell cycle or apoptosis analysis.

Reagents

RNase Preparation

  • 10mM Tris hydrochloride + 15mM NaCl + RNase to make final dilution of 10mg/ml
  • Put tube of this solution in boiling H2O for 10 minutes to deactivate DNase

Propidium Iodide Solution

  • 100µg/ml in PBS

Staining

  1. Pellet 1 x 106 cells.
  2. Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
  3. Resuspend cells in 100µl PBS.
  4. Add 3mls of -20°C 70% EtOH (ethanol).
  5. Mix.
  6. Incubate 1 hour at 4°C.
  7. Wash cells twice with 2mls PBS each time. Carefully aspirate supernatant after each centrifuge step so that you don't lose cells.
  8. Resuspend in 100µl PBS.
  9. Dilute stock RNase A solution 1:10 and add 100µl to cell suspension.
  10. Add 200µl PI solution to cell suspension.
  11. Mix.
  12. Incubate at 4°C for 30 minutes in the dark.
  13. Run on flow cytometer.