Fitc conjugated anti mouse alexa fluor 488 secondary antibody

BJ cells were seeded in six-well plates on day 1. The next morning, cells were incubated in fresh medium for 3 h with 30 μM bromodeoxyuridine (BrdU, Sigma) followed by two washes with phosphate-buffered saline (PBS) and then fixed with 4% formaldehyde. Cells were washed twice with PBS and treated with 5 M HCl/0.5% Triton to denature DNA. Cells were neutralized with 0.1 M Na₂B₄O₇. The cells were then treated with blocking buffer (3% BSA in 0.5% Tween PBS) for 30 min and incubated with anti-BrdU antibody (Dako) with blocking buffer for 2 h at room temperature. Cells were washed with PBS three times and finally incubated with FITC-conjugated anti-mouse Alexa Fluor 488 secondary antibody (Dako) in blocking buffer for 1 h, washed three times, and stained with propidium iodide for 30 min. BrdU incorporation was measured by immunofluorescence (at least 1000 cells were scored for each condition). The numbers of individual nuclei and BrdU-stained nuclei were counted using imageJ software.

For PC3 cells, a final concentration of 10 μM bromodeoxyuridine (BrdU, Sigma) was added to the medium and incubated for 25 min. Cells were harvested and fixed with 70% cold ethanol at 4°C for 30 min. RNase A treatment (final concentration at 0.5 mg/ml) at 37°C for 30 min was applied. Cells were resuspended in freshly prepared HCl/0.5% Triton solution (for DNA denature) at room temperature for 20 min and then neutralized by 0.1 M Na₂B₄O₇. After washed once with PBS/Tween, cells were incubated with 1:40 diluted anti‐BrdU antibody (Dako) at RT for 30 min. Cells were incubated with FITC‐conjugated anti‐mouse Alexa Fluor 488 secondary antibody (1:500, Dako) at RT for 30 min in the dark. After washing another 2X, cells were then resuspended in PI (20 μg/ml) solution and ready for FACS assay (at least 10,000 cells were gated for each condition).