Cell Cycle – DNA Content
- 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 4o C.
- 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.
|Stock Propidium Iodide (4.5 mg/ml)||33 ul|
|RNase A (20 mg/ml)||400 ul|
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.