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Star Republic: Guide for Biologists

BrdU labeling of S phase cells

Bromodeoxyuridine (BrdU) is an analogue of thymidine and will be taken up into the DNA of cells in the S phase. BrdU is detected using an antibody against BrdU. The following protocol is for FACS analysis. For histochemical staining, cells should be left on plates or class slides (omit step 2).

1. Label cells with BrdU at the final concentration of 10 mM for 30 minutes to 24 hours.

2. Scrape cells into the medium. Centrifuge at 1000 rpm for 5 minutes to pellet cells.

3. Wash the cell pellets with cold PBS. Centrifuge at 1000 rpm for 5 minutes to pellet cells.

4. Fix cells in cold 70% ethanol for 30 minutes at 4 C. Vortex to prevent clumping of cells. Cells can be stored at 4 C at this stage.

5. Spin down cells and wash cells twice with PBS.

6. Resuspend cell pellets in 2 M hydrochloric acid (1 volume concentrated hydrochloric acid mixed with 5 volumes water). Unwind DNA at room temperature for 30 minutes.

7. Spin down cells and wash three times with PBS/0.2%-Tween 20.

8. Add 200 ml anti-BrdU antibody (1:100) to cells and incubate for 20 minutes at room temperature.

9. Spin down cells and wash three times with PBS/0.2%-Tween 20.

10. Add 50 l anti-mouse IgG-FTIC (1:10) and incubate for 20 minutes at room temperature in the dark.

11. Spin down cells and wash once with PBS.

12. Treat with 50 ml ribonuclease (100 mg/ml) for 15 minutes at room temperature.

13. Stain DNA with 200 ml propidium iodide (50 mg/ml). Incubate for at least 30 minutes.

14. Analyse by FACS.