For adherent cells in 100mm plate2
- Transfer media to 50mL centrifuge tube
- Wash plate with cold PBS and transfer to 50mL tube from above
- Trypsinize cells and deactivate trypsin with media containing serum and transfer to 50mL centrifuge tube from above.
- Fill 50mL centrifuge tube with cold PBS.
- Spin at 1500rpm for 10 minutes.
- There should be a pellet
- Remove media and resuspend by slowly adding 1mL ice-cold 70% ethanol while vortexing.
- Store at -20oC for overnight, not more than 1 week.
When ready to analyze
- Spin cells fixed in ethanol at 2000rpm for 15 minutes.
- Remove ethanol
- Resuspend cells in 0.5mL cold PBS for small pellet and 1 mL cold PBS for large pellet and transfer to flow cytometer friendly tube
- Add 1/20 volume of 10mg/mL RNAse A (in TE buffer)
- Add 1/40 volume of 1.6mg/mL propidium iodide (in ddH2o)
- Incubate at 37oC for 30 minutes covered
- Put samples on ice, covered.
- Analyze using flow cytometry within an hour.
Protocol adapted by: T. Landowski, Ph.D., and Alan Pourpak