BrdU Staining of Cells with Rabbit Anti-BrdU Antibody by Flow Cytometry

Reagents

0.05% (v/v) Tween 20 in phosphate buffered saline (PBS)

0.1 M Na₂B₄O₇, pH 8.5

2 M hydrochloric acid (HCl) containing 0.5% Triton X-100

DNA stains/cell viability dyes, such as propidium iodide

Rabbit Anti-BrdU Antibody (AHP2405)

PBS

PBS containing 1% bovine serum albumin (PBS/BSA)

Methods

1. Label cells with BrdU. Add BrdU to the cell suspension in culture medium to a final concentration of 10 µM and incubate for 30 min in a CO₂ incubator at 37°C.
2. Wash cells 2 times (2x) with PBS/BSA, centrifuge cells at 500 x g for 10 min, decant supernatant, and resuspend in a minimum volume of PBS.
3. Add cells slowly into 5 ml 70% ethanol chilled to -20°C, mix continuously by vortexing.
4. Incubate on ice for 30 min.
5. Centrifuge at 500 x g for 10 min, and decant supernatant.
6. Add 2 ml of 2 M HCl containing 0.5% Triton X-100, and incubate the cells for 30 min at room temperature (RT) on a rocking platform set to 15 rpm. Denaturation of the DNA by HCl is critical for successful BrdU staining. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend in 1 ml of 0.1 M Na₂B₄O₇,
   pH 8.5.
7. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend the cells in PBS/BSA plus 0.05 % Tween 20 to a concentration of 1 x 10⁷ cells/ml.
8. Aliquot 100 µl of the cell suspension into the required number of FACS tubes.
9. Incubate cells with Rabbit Anti-BrdU Antibody at the recommended dilution for 1 hr at RT. Alternatively, incubation can be performed overnight at 4°C.
10. To wash, add 2 ml of PBS/BSA, and centrifuge the cells at 500 x g for 5 min.
11. Perform incubation with a secondary antibody, such as Sheep Anti-Rabbit IgG DyLight 488 Conjugated Secondary Antibody (STAR36D488GA). For this purpose decant the supernatant and perform secondary antibody incubation for 45 min at RT (refer to the respective datasheet about the recommended dilution). Wash cells by repeating step 10.
12. Decant the supernatant and add 0.5 ml of sheath fluid.
13. For total DNA staining, add DNA stains such as, ReadiDrop™ Propidium Iodide (1351101),ReadiDrop 7-AAD (1351102), or PureBlu™ Hoechst 33342 (1351304), to each tube. 
14. Analyze cells by flow cytometry following the manufacturer’s instructions. The DNA stain should be read on the appropriate channel set to the height and linear scale and not log scale.

Notes

The acid treatment to unwind the DNA may affect surface immunophenotyping. Staining of cells with BrdU using DNAse I may be applicable if this is required.

Appropriate controls should be carried out for flow cytometry, consider including the following:

• A known positive sample
• Isotype controls (to determine if the staining is specific)
• Unstained cells (should always be included to monitor autofluorescence)

For all multicolor flow cytometry experiments include compensation controls and fluorescence minus one (FMO) controls, which assist with identifying gating boundaries.