**These protocols are for staining the DNA of all cells in a sample for use in DNA cell cycle analysis. They are not recommended for apoptosis analysis, except as a preliminary check for DNA degradation (subG0). They are also not appropriate to check viability, since the samples are fixed in EtOH prior to staining with PI.**
Step 1: Harvest and count cells.
Step 2: Centrifuge and remove supernatant. Vigorously vortex the pellet for 10 seconds and continue to vortex the cells while slowly adding 1 ml of ice cold 70% ethanol drop by drop to the pellet. Fix overnight at 4 o C.
Step 3: Prepare PI staining solution (prepare fresh for each staining assay).
-8.5 ml staining buffer (0.1% BSA in PBS)
-1 ml Rnase (stock at 1% – 1:10 = 0.1%)
-0.5 ml PI stock solution (stock at 1 mg/ml)
Step 4: Briefly vortex tube with sample. Centrifuge 10 minutes at high speed (3000 rpm) and remove tube. Pour off ethanol. Leave less than or equal to 0.2 ml ethanol in tube.
Step 5: Gently vortex tube to suspend cells in residual ethanol. Add 0.5 to 1.0 ml PI staining solution to each tube and vortex. Let sample incubate for greater than or equal to 30 minutes at room temperature in the dark.
Step 6: Transfer to 5 ml collection tubes (12 x 75 mm), if necessary, and analyze on the Fortessa in the UAMS Flow Cytometry Core Facility.