Anti - BrdU Staining Using 70 % Ethanol and 2N HCl
Pulse actively dividing cells with BrdU ( in vitro , cell culture media can be pulsed by adding 10 - 40 μM of BrdU for 1 - 2 hours ) .
Harvest cells and centrifuge for 5 minutes at 1200 - 1500 rpm ( 200 - 300 x g )
Wash cells in 1x PBS ( PBS , 10x Concentrate , Cat . No . 926201 ) and centrifuge for 5 minutes at 1200 - 1500 rpm ( 200 - 300 x g ) .
Discard supernatant .
NOTE : The combined presence of proteins and HCl in downstream steps may cause aggregation .
As such , it is highly recommended that wash steps utilize PBS without any protein additive until otherwise indicated .
Dislodge cell pellet and add 5 ml of ice - cold ( - 20⁰C ) 70 % Ethanol to 1 - 2 X 107 cells dropwise while slowly vortexing .
Incubate at - 20⁰C for at least 2 hours .
Cells may be stored for several days .
Repeat step 3 twice .
Dislodge cell pellet and add 2 ml of 2 N HCl and incubate for 20 minutes at room temperature
Repeat step 3 .
Resuspend cells at a concentration of 1 x 107 cells per / ml of staining buffer and aliquot 100 μl per tube .
Add anti - BrdU antibody at appropriate concentration and incubate for 20 minutes at room temperature .
Wash cells in Cell Staining Buffer ( Cat . No . 420201 ) and centrifuge for 5 minutes at 1200 - 1500 rpm ( 200 - 300 x g ) .
Stain DNA by adding 1 μg of either 7 - AAD ( Cat . No . 420403 ) or DAPI ( Cat . No . 422801 ) .
Wait for 5 minutes prior to acquiring samples on flow cytometer
