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 .
