70% Ethanol at – 20oC
DNA Staining Buffer:
Sodium citrate 0.25g
Triton–x 100 0.75ml
Propidium iodide 0.025g
Ribonuclease A 0.005g
Distilled water 250 ml
1. Place 1x106 cells from each sample into a polypropylene tube and centrifuge at 250 x g for 5 min.
2. Remove the supernatant as completely as possible without disturbing the pellet and add 1 ml of –20oC 70% EtOH dropwise to the cell pellet while vortexing.
3. Keep cells at -20oC until the day of DNA staining (cells can be stored for several weeks at -20oC).
4. On the day of DNA staining, take samples out of the freezer and spin them down by centrifugation at 250 x g for 5 min. Remove the supernatant as completely as possible without disturbing the cell pellet.
5. Add 1 ml of DNA staining buffer to the cell pellet and vortex gently and briefly. Keep cells for 15 min in the staining solution before acquisition on the flow cytometer.
Sodium citrate Cat# C7254 Sigma, St. Louis, MO
Triton-x 100 Cat# x-100 "
Ribonuclease A Cat# R4875 "
Propidium iodide Cat# 537059 Calbiochem, San Diego, CA