BrdU cell proliferation assay

BrdU assay was performed using BrdU cell proliferation assay kit (Cell Signaling, Beverly, MA) according to manufacturer’s instructions. Briefly, 5000 cells were seeded in a 96-well plate and incubated overnight followed by adding 10 µM BrdU and incubation for 12 hr. After the medium was removed, cells were fixed by 100 µl/well of fixing solution for 30 min at room temperature. BrdU was detected by 100 µl/well of 1X detection antibody solution followed by 1X HRP-conjugated secondary antibody solution. Then, 100 µl TMB substrate was added to each well and incubated at room temperature for 30 min followed by 100 µl of stop buffer. The absorbance was read at 450 nm using a plate spectrophotometer.