BrdU Pulse

1. Plate 300,000 cells per well in 6-well dishes and incubate overnight at 37'C
2. Pulse cells with 10uM BrdU in culture media for 30 min. at 37'C
3. Wash cells 2 x PBS and add fresh media
4. Treat experimental cells with 10 Gy ionizing radiation

Fix Cells

1. Harvest cells (I use 500 uL 0.5% trypsin per well) at time points following treatment and transfer to microcentrifuge tubes
2. Centrufuge at 10,000 rpm for 10 sec. and aspirate supernatant
3. Add 150 uL cold PBS to cells and pipet up and down to mix.
4. Add 350 uL cold 100% MeOH giving a final concentration of 70% MeOH
5. Incubate fixed cells overnight at -20'C

BrdU and PI staining

1. Centrifuge fixed cells at 10,000 rpm for 10 sec. and aspirate supernatant
2. Add 250 mL of 2 N HCL and 0.2 mg/mL pepsin and incubate at room temp. for EXACTLY 30 min.
3. Add 750 mL 0.1 M sodium tetraborate (pH 8.5) to neutralize HCL
4. Centrifuge at 10,000 rpm for 10 sec. and aspirate supernatant
5. Wash cells with 1% BSA in PBS
6. Centrifuge at 10,000 rpm for 10 sec. and aspirate supernatant
7. Wash with 1% BSA/0.5% Tween-20 in PBS, centrifuge as above and aspirate supernatant
8. Add 50 uL of antibody (1:20 dilution of Alexafluor 647-conjugated anti-BrdU antibody in 1% BSA/0.5% Tween-20 in PBS)
9. Incubate at room temp. in the DARK for 30 min.
10. Wash with 500 uL PBS, centrifuge and aspirate as before
11. Add 400 uL PI solution (1/10 volume 1 mg/mL propidium iodide, 1/10 volume 10 mg/mL RNase A, 8/10 volume PBS) to each pellet and transfer to flow tube
12. Incubate in DARK at 37'C for 30 min.
13. Run samples on FACS Calibur