Anti-BrdU Staining Protocol

1. Pulse BrdU: Make sure cultured cells are in log growth phase prior to pulsing with BrdU - change the cell culture medium one day before use. Do not wash cells prior to incubation with BrdU - add BrdU directly to the culture medium to achieve a final BrdU concentration of 10 µM. Incubate the cells 1 hour in CO2 incubator at 37°C.

2. Harvest cells and spin at 1000 rpm for 5 min and wash 1X with PBS.3. Add 5 ml 70% ethanol (pre-chilled at -20 ° C) to cell pellet drop by drop while vor-

texing, incubate at room temperature for 20 min.4. Wash 1X with PBS.5. Resuspend cells pellet in 2 ml 2N HCL and incubate at room temperature for 20

min.6. Wash 1X with PBS.7. Resuspend cell pellet in 2 ml of 0.1 M Na2B4O7 for 2 min.8. Wash 1X with cell staining buffer.9. Resuspend cells in cell staining buffer at the cell concentration of 107/ml for direct

or indirect immunofluorescence staining.10. After staining, resuspend cells in either PI or 7-AAD buffer and analyze data in

combination with DNA analysis.

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References:

Pozarowski, P. and Darzynkiewicz, Z. 2004. Methods Mol. Biol. 281:301.