Flow Cytometric Analysis of Cell Cycle
Fixation
1) Collect 2 X 106 cells.
2) Pellet cells by spinning at 1,000 rpm, 4°C for 5 minutes.
3) Resuspend cell pellet in 1 ml of cold PBS.
4) Fix cells by adding 4 ml of -20°C absolute ethanol.
5) Store cells at -20°C in this fixation buffer until ready for analysis. (No more than 2 weeks)
Staining
6) Centrifuge (as above) fixed cells and resuspend pellet in 1 ml of PBS.
7) Add 100 µl of 200 µg/ml DNase-free, RNaseA and incubate at 37°C for 30 minutes.
8) Add 100 µl of 1 mg/ml propidium iodide (light sensitive) and incubate at room temperature for 5-10 minutes.
9) Place samples in 12 X 75 Falcon tubes and read on Becton Dickinson FACStarPLUS.
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