Anti-BrdU Staining Protocols Using DNAse with Surface and Fluorescent Proteins
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 Cell Staining Buffer (Cat. No. 420201) and centrifuge for 5 minutes at 1200-1500 rpm (200-300 x g).
Discard supernatant. 
Aliquot 5 x 105- 1 x 106 cells per 12 x 75 mm tube. 
Optional: Stain cells for surface antigens if required, utilizing the Cell Surface Immunofluorescence Staining Protocol (see link). 
Wash cells by adding 1 ml of Cell Staining Buffer to each tube and centrifuging for 5 minutes at 1200-1500 rpm (200-300 x g).
Discard supernatant. 
Fix cells by adding 100 μl of 4% paraformaldehyde at room temperature for 20-30 minutes. 
Wash cells by repeating step 6 twice.
(Optional: Cells can be stored in FACS buffer at 4⁰C for up to 72 hrs).
Permeabilize cells by adding 500 μl of 0.5% Triton-X 100 in PBS for 15 minutes at room temperature. 
Wash cells by repeating step 6 twice. 
Treat cells with 20 μg of DNAse (Cat. No. D4513, Sigma-Aldrich) diluted in DPBS with calcium and magnesium to each tube and incubate at 37⁰C for 1 hour.
Wash cells by repeating step 6 twice. 
Add 50 μl of Cell Staining Buffer to each tube then add the recommended concentration of anti-BrdU antibody to each tube.
Incubate for 20 minutes at room temperature in the dark Repeat step 6.
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.
