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 M. Incubate the cells 1 hour in CO2 incubator at
37C.
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 vortexing, 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.

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

9727 Pacific Heights Blvd. San Diego, CA 92121

Phone: 858-455-9588 Toll Free: 1-877-246-5343 Fax: 858-455-9587
www.biolegend.com
Rev. 080312