BrdU Incorporation Assay for Cell Proliferation

For the estimation of cell proliferation, cells were allowed to attach on glass coverslips before labeling with 50 μM BrdU for 48 h, as reported before [19 (link),41 (link),42 (link)]. Cells were then fixed with 4% (v/v) formaldehyde in PBS for 10 min, permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 min and denaturation of DNA was achieved after treatment with 2 N HCl for 30 min. After blocking with 0.5% (v/v) gelatin in PBS, samples were incubated with an anti-BrdU-FITC antibody purchased from BioLegend (SanDiego, CA, USA) at 4 °C and counterstained with 2 μg/mL DAPI in PBS for 20 min. The percentage of cells with a proliferative potential in a given cell population was calculated by dividing the number of BrdU-positive nuclei by the number of the DAPI-positive nuclei, as counted under a Zeiss Axioplan 2 fluorescent microscope.