Stewart Laboratory Standard Operating Procedure               

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BrdU Incorporation Protocol

Day 1

1. Label cells with media containing100 μM BrdU (6 μL/mL of 5 mg/mL stock):

• Diploid Fibroblasts, 4-6 hours
• ES cells, 30 minutes
• 3T3 cells, 2.5 hours

1. Trypsinize cells, wash from plate with PBS, spin down (400 x g, 1700 rpm, 5 min in 15 ml tubes - no faster!), and resuspend in 10 mL 70% cold EtOH.
2. 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 resuspend in 0.5 mL 2M HCl + 0.5% IFS (make fresh!). Incubate at RT for 20 minutes.
3. Wash cells once with 1 mL wash buffer.
4. Pellet and resuspend in 0.1 M sodium tetraborate (Na₂ B₄ O₇ ). Incubate at RT for 2 minutes.
5. _{Wash cells twice with 1 mL wash buffer (if a negative control of PI alone stain is desired, set aside a fraction of cells until step 10).}
6. _{Resuspend cell pellet in anti-BrdU antibody (50 μL total solution diluted with wash buffer). Incubate for 20 minutes at 4°C.}

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

Or anti-BrdU: Boeringher #1170376 1:10 dilution

1. Wash cells once with 1.5 mL wash buffer.
2. Resuspend cell pellet in anti-mouse FITC antibody (50 μL total solution with wash buffer, 1:500 dilution). Incubate for 20 minutes at 4°C.
3. Wash cells once with 1.5 mL wash buffer.
4. Resuspend pellet in 0.3 mL PI (10 μg/mL in wash buffer). Incubate for 30 minutes at RT.
5. Cells are ready for analysis by flow cytometry.