Cell Cycle with PI

 

For adherent cells in 100mm plate2

  1. Transfer media to 50mL centrifuge tube
  2. Wash plate with cold PBS and transfer to 50mL tube from above
  3. Trypsinize cells and deactivate trypsin with media containing serum and transfer to 50mL centrifuge tube from above.
  4. Fill 50mL centrifuge tube with cold PBS.
  5. Spin at 1500rpm for 10 minutes.
  6. There should be a pellet
  7. Remove media and resuspend by slowly adding 1mL ice-cold 70% ethanol while vortexing.
  8. Store at -20oC for overnight, not more than 1 week.

When ready to analyze

  1. Spin cells fixed in ethanol at 2000rpm for 15 minutes.
  2. Remove ethanol
  3. 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
  4. Add 1/20 volume of 10mg/mL RNAse A (in TE buffer)
  5. Add 1/40 volume of 1.6mg/mL propidium iodide (in ddH2o)
  6. Incubate at 37oC for 30 minutes covered
  7. Put samples on ice, covered.
  8. Analyze using flow cytometry within an hour.

Protocol adapted by: T. Landowski, Ph.D., and Alan Pourpak