1) Collect 1 X 106 cells after proper treatment.
2) Pellet cells by spinning at 2,000 rpm, 4C for 5 minutes.
3) Resuspend cell pellet in 1 ml of cold FCM buffer (5mM EDTA in 1xPBS) .
4) Fix cells by adding 1 ml of -20C absolute ethanol.
5) Store cells at -20C in this fixation buffer until ready for analysis. (No more than 2 weeks)
6) Centrifuge (as above) fixed cells and resuspend pellet in 1 ml of FCM buffer.
7) Add 2 ul mg/ml DNase-free, RNaseA and incubate at 37C for 20 minutes.
8) Add 1 ul of 5 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 15 minutes.
9) Pellet the cells by spinning at 2,000 rpm, 4C for 5 minutes.
10) Wash with FCM buffer once more, discard the buffer and resuspend the stained cells with 500ul FCM buffer.
11) Place samples in 12 X 75 Falcon tubes and read the data (% of G0 G1 G2 MS) on a FACS analysis system.