Anti brdu primary antibody

For the BrdU (5-bromo-2′-deoxyuridine) incorporation assay, cells were cultured with a BrdU-labeling reagent (Life Technologies), performed DNA hydrolysis with 1 M HCl, and stained with an anti-BrdU primary antibody (Cell Signaling) and Alexa Fluor^® 594 secondary antibody according to the manufacturer’s instructions.

Cells were plated in six-well plates and 24 h later washed with fresh medium only, followed by treatment with fresh medium containing the appropriate drug concentrations. One hour prior to harvest, cells were incubated with 10 μM BrdU (Thermo Fisher Scientific, Loughborough, UK) before fixing with 70% (v/v) ethanol/PBS at 4 °C for at least 10 min. Cells were centrifuged (500 × g, 4 °C, 5 min) and washed twice in PBS, DNA denatured with 1.5 M HCl for 30 min and again centrifuged (500 × g, 4 °C, 5 min) and washed twice in PBS. Cells were then blocked in 0.5% (w/v) BSA/PBS for 10 min at RT before addition of anti-BrdU primary antibody (Cell Signaling Technology, NEB, Hitchin, UK) diluted 1:200 for 1 h at RT. Following centrifugation (500 × g, 4 °C, 5 min) and washing twice in 0.5% (w/v) BSA/PBS, cells were incubated with anti-mouse fluorochrome-conjugated secondary antibody (anti-mouse Alexa Fluor 488, Thermo Fisher Scientific, Loughborough, UK) diluted 1:200 for 30 min at RT. Cells were then centrifuged (500 × g, 4 °C, 5 min) and washed in 0.5% (w/v) BSA/PBS three times, and finally resuspended in 0.2 mL PBS. BrdU-positive cells were assessed with a FACS LSRII flow cytometer (BD Biosciences, Oxford, UK), counting 10,000 cells per sample using no BrdU samples as gating controls. Data were analyzed using FlowJo software (FlowJo LLC, Oregon, USA).