The University of Iowa Carver College of Medicine
Whole Cell DNA Staining
The following protocol is for whole cell DNA staining with propidium iodide for cell cycle or apoptosis analysis.
- 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
- Pellet 1 x 106 cells.
- Carefully aspirate supernatant leaving as little buffer as possible without aspirating cells.
- Resuspend cells in 100µl PBS.
- Add 3mls of -20°C 70% EtOH (ethanol).
- Incubate 1 hour at 4°C.
- Wash cells twice with 2mls PBS each time. Carefully aspirate supernatant after each centrifuge step so that you don't lose cells.
- Resuspend in 100µl PBS.
- Dilute stock RNase A solution 1:10 and add 100µl to cell suspension.
- Add 200µl PI solution to cell suspension.
- Incubate at 4°C for 30 minutes in the dark.
- Run on flow cytometer.