APC BrdU Flow Kit

Protocol tips

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	- Cells are treated with 10 μm bromodeoxyuridine (BrdU) for 1 h. - Cells are fixed, and permeabilized with BD Cytofix/ Cytoperm Buffer containing 3–5% formaldehyde.

Protocol tips
- Cells are treated with 10 μm bromodeoxyuridine (BrdU) for 1 h. - Cells are fixed, and permeabilized with BD Cytofix/ Cytoperm Buffer containing 3–5% formaldehyde.

Publication protocol

The cell cycle analysis was performed using APC BrdU flow kit (BD Biosciences) as described elsewhere (34). Briefly, cultured cells were labeled with 10 μm bromodeoxyuridine (BrdU) for 1 h, washed, collected, fixed, and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) containing 3–5% formaldehyde. Then the cells were treated with DNase (300 μg/ml) to expose BrdU epitope for 1 h at 37 °C, washed, and then stained with FITC-conjugated anti-BrdU antibody for 20 min at room temperature, washed again, and centrifuged. Staining buffer containing 7-aminoactinomycin D (7-AAD) was added to each tube to resuspend the cells, and the stained cells were analyzed by flow cytometry using FACScan II (BD Biosciences). Acquired multiparameter data were analyzed using CellQuest software (BD Biosciences) with the combination of BrdU and 7-AAD; two color flow cytometric analysis permits the enumeration and characterization of cells that are actively synthesizing DNA (BrdU incorporation) in terms of their cell cycle position. As shown by the region gates applied to the 7-AAD versus BrdU dot plot, flow cytometric analysis of cells stained with the reagents allowed the discrimination of cell subsets that were in the S phase (green), G0/G1 phase (purple), and G2+M phase (blue).

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