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.
