（1）The protocol for DNA staining by propidium iodide （PI）is as follows:
1. Harvest cells in the appropriate manner and wash in 1ml PBS. 2. Add 3ml cold 100% ethanol. Add dropwise to the cell pellet while vortexing. This should ensure fixation of all cells and minimise clumping. 3. Fix for at least 30 minutes at 4°C. Specimens can be left at this stage for several weeks. 4. Wash x2 in PBS. Spin at 2000rpm and be careful to avoid cell loss when discarding supernatant especially after spinning out of ethanol. 5. To ensure that only DNA is stained, treat cells with Ribonuclease. Add 50 µl of 100 µg/ml RNase. 6. Add 200 µl propidium iodide (50 µg/ml) Or final concentration 15 umol/L. 7. Analyse by flow cytometry
（2）BROMODEOXYURIDINE (BrdU) STAINING
1. Treat cells with 10 µM BrdU for an appropriate time (30 minutes - 48 hours). 2. Harvest cells, wash. 3. Fix cells in cold (4°C) 70% ethanol. Vortex while fixing to prevent excess clumping and to ensure adequate fixation of all cells. Leave for at least 30 minutes at 4°C. Samples may be left at this stage for up to a week (at least). AT THIS STAGE, SAMPLES SHOULD BE BROUGHT TO THE FACS LAB FOR PROCESSING. 4. Spin off alcohol (2000 rpm, 5 minutes). Wash x2 in PBS. 5. Re-suspend cells in 2M hydrochloric acid (made from 1 part Conc HCl to 4 parts distilled water). Leave at room temperature for 30 minutes, mixing at intervals. 6. Spin off acid. 7. Wash x2 in PBS and x1 in PBS-T (PBS + 0.1% BSA + 0.2% Tween 20, pH 7.4). 8. Add 2 µl anti-BrdU antibody (Becton Dickinson) directly to the cell pellet and incubate for 20 minutes at room temperature in the dark. 9. Wash x2 in PBS-T. 10. Stain with 50 µl FITC-conjugated rabbit anti-mouse F(ab')2 fragments (DAKO) at 1 in 10 for 20 minutes at room temperature in the dark. 11. Wash x1 in PBS. 12. Treat with 50 µl ribonuclease (100 µg/ml. Sigma) for 15 minutes at room temperature. 13. Add 200 µl propidium iodide (50 µg/ml). Leave for at least 30 minutes. 14. Analyse by flow cytometry. Fluorochromes are excited by a 488nm laser. FITC fluorescence is collected between 515 and 545nm and propidium fluorescence above 580nm. Forward and right angle scatter are used to define the cellular population and pulse processing of the propidium signal is used to distinguish true G2 cells from G1 doublets. 20,000 events are normally acquired.