Analysis of Cell Cycle Based on DNA Content FIXATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS OF DNA BY PI STAINING R. C. Bird                                                                                                                                 FIXATION Liberate cells from tissue culture flask by trypsin digestion and collect in a 15 ml conical tube. Collect cells by centrifugation 3000xg 4 min and resuspend in 1 ml PBS. Repeat PBS wash. Add 3 ml 70% ethanol (-20oC) slowly DROP-WISE directly from the freezer while vortexing VERY GENTLY (No. 2 vortex setting on mixer). Incubate on ice for 30 min Collect cells by centrifugation 3000xg 2 min and resuspend in 1 ml Flow Buffer or PBS. Adjust volume to adjust the cell concentration. Store tightly capped at 4oC. STAINING At least 30 min before analysis begins stain the cells. Combine 0.1 ml of cells with 0.9 ml stain solution. Incubate 30 min (or longer) at room temperature in the dark. Stained samples may be stored in the dark at 4oC. STAINING SOLUTION                                                                     10 ml   Stock Propidium Iodide              (4.5 mg/ml)                                        33 ul RNase A                                  (20 mg/ml)                                            400 ul Water                                                                                                   9.57 ml Reference for this method: You J, Bird RC. Selective induction of cell cycle regulatory genes cdk1 (p34cdc2), cyclins A/B, and the tumor suppressor gene Rb in transformed cells by okadaic acid.J Cell Physiol. 1995 Aug;164(2):424-33.