The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Whole Cell DNA Staining

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.