Labeling of Cells with BrdU:
Add BrdU (Sigma) at a final concentration of 10 uM to approximately 1 x 106 cells, and incubate under the appropriate growth conditions for 15 to 60 minutes to pulse label the cells. Times may vary depending on the cell line.
Collecting the Cells:
Scrape or trypsinize the cells if using adherent cells. If you are interested in analyzing cell death, collect cells floating in the media as well as the adherent population.
Pellet the cells at 800 to 1000 rpm for 5 to 10 minutes and remove the supernatant.
Wash the pellet in 5 ml of 1X cold PBS and remove the supernatant.
Resuspend the pellet gently in 100 ul of cold PBS and keep the cells on ice.
Fix the cells by dropping them slowly into 5 ml of ice cold Ethanol while maintaining a Gentle vortex.
Place the cells at 4°C for at least 30 minutes or at 4°C overnight, which is the preferred method for optimal fixation.