Reagents

  • 0.5% Paraformaldehyde
  • 1x PBS
  • 70% methanol
    • cooled to -20°C or -80°C (either will work)
  • RNAse A
    • 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
    • 100µg/ml in PBS

Staining

  1. Pellet 1 x 106 cells in a microfuge tube.
  2. Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
  3. Resuspend cells in 0.5% paraformaldehyde for 15 minutes at 4°C.
  4. Wash cells twice with 2ml PBS each time leaving 30µl of volume in the bottom of the microfuge tube. Carefully aspirate supernatant after each centrifuge step so that you don't lose cells.
  5. Vortex and immediately add 1ml of -20°C or -80°C 70% methanol.
  6. Immediately cap the tube and vortex to prevent clumping.
  7. Incubate 1 hour at 4°C.
  8. Wash cells once with 2ml PBS. Carefully aspirate supernatant so that you don't lose cells.
  9. Resuspend in 100µl PBS.
  10. Dilute stock RNAase A solution 1:10 and add 100µl to cell suspension.
  11. Add 200µl PI solution to cell suspension.
  12. Vortex.
  13. Incubate at 4°C for 30 minutes in the dark.
  14. Transfer cell preparation to test tubes compatible with flow cytometer.
  15. Run on flow cytometer.