STEWART LABORATORY

BrdU INCORPORATION PROTOCOL

Day 1

1. Label cells with media containing 100 µM BrdU (6 µL/mL of 5 mg/mL stock):

Ø ES cells, 30 minutes

Ø 3T3 cells, 2.5 hours

Ø Diploid Fibroblasts, 4-6 hours

2. Trypsinize cells, wash from plate with PBS, spin down (400 x g, 1700 rpm, 5 min in 15 mL tubes – no faster!) and re-suspend in 10 mL
70% EtOH

3. Store at -20°C overnight or for several days

Day 2

1. Pellet cells and wash once with wash buffer (PBS + 0.5% IFS)

2. Pellet and re-suspend in 0.5 mL 2M HCl + 0.5% IFS (make fresh!). Incubate at RT for 20 min

3. Wash cells once with 1 mL wash buffer

4. Pellet and re-suspend in 0.1M sodium tetraborate (Na₂B₄O₇). Incubate at RT for 2 min

5. Wash cells x2 with 1 mL wash buffer (set aside a fraction of cells that will be stained with only PI—these will be used in step 10)

6. Re-suspend cell pellet in anti-BrdU antibody (50µL total solution diluted with wash buffer). Incubate for 20 min at 4°C

Use BrdU-FITC: Boeringher # 1202693 1:100 dilution (skip to step 9)

OR

anti-BrdU: Boeringher # 1170376 1:10 dilution

7. Wash cells once with 1.5 mL wash buffer

8. Re-suspend cell pellet in anti-mouse FITC antibody (50µL total solution diluted with wash buffer, 1:500 dilution). Incubate for 20 min
at 4°C

9. Wash cells x1 with 1.5 mL wash buffer

10. Re-suspend pellet in 0.3 mL PI (10 µg/mL in wash buffer). Incubate for 30 min at RT

11. Cells are ready for analysis by flow cytometry