Bromodeoxyuridine (BrdU) Staining Protocol

1. Incubate cells in culture with 10 mM BrdU (Sigma) for an appropriate time (30 mins – hour plus)
2. Harvest cells.
3. Fix pelleted cells in ice-cold 70% ethanol by adding with a pasteur pipette on a vortex. Leave cells at 4^oC from 30 mins to a week.
4. Pellet cells at approximately 2,000 rpm for 5 mins. Wash twice in PBS.
5. Res-suspend cells in freshly made 2M HCl (dilute conc HCl 1 part to 4 parts distilled water. Leave for 30 mins at RT with occasional mixing.
6. Pellet cells, wash twice in PBS.
7. Wash in PBS-Tween (PBS+0.1% BSA+0.2% Tween20, pH7.4).
8. Add 2 ml anti-BrdU mcab (Becton Dickinson) to the cell pellet and incubate at RT for 20 mins in the dark as BrdU is photo-unstable.
9. Wash twice in PBS-Tween.
10. Add 5 ml of rabbit anti-mouse^FITC F(ab^’)₂ fragments (DAKO, Cat.No.F0313) to the cell pellet and incubate at RT for 20 mins.
11. Wash in PBS.
12. Add 50 ml RNAse (100 mg/ml Sigma) and incubate at RT or 37^oC for 15 mins.
13. Add 200 ml of PI (50 mg/ml Sigma P4170).
14. Analyse by flow cytometry collecting 25,000 events per sample.