Brdu cell proliferation chemiluminescent assay kit

Cell proliferation assay was performed in a 96-well plate. Proliferation assays were carried out using BrdU Cell Proliferation Chemiluminescent Assay Kit (#5492, Cell Signaling). LNCaP and PC-3 cells were incubated with BrdU for 4 hours and 2 hours respectively. Chemiluminescence was determined using a Multiskan™ FC Microplate Photometer (Thermo Scientific).

Measurement of cell proliferation was achieved by using the BrdU Cell Proliferation Chemiluminescent Assay Kit (Cell Signalling Technology) following the supplier's instruction. Cells were seeded at a density of 5 × 10⁴ cells/well and incubated for 24 h prior to exposure. Post exposure, the complete media was replaced with BrdU solution diluted in complete media (1/1000 dilution) and the cells returned to the incubator for 48 h to allow for BrdU incorporation. Subsequently, cells were fixed in 4% PFA for 15 min, washed in PBS and denatured (Cell Signalling Technology). After washing twice in PBS, anti-BrdU antibody (Cell Signalling Technology, 1/100 dilution) was added and incubated for 1 h at room temperature. Cells were then washed and blocked with 5% fetal calf serum for 45 min prior to adding anti-mouse 647 secondary antibody (Life Technologies, 1/400 dilution) and incubating for 1 h at room temperature. Finally, the cells were washed twice in PBS and imaged.

To examine the effect of atezolizumab on MDA-MB-231 cell proliferation, a BrdU cell proliferation chemiluminescent assay kit was utilized (Cell Signaling Technology). This assay detects BrdU incorporation into cellular DNA during cell proliferation. On day 0, MDA-MB-231 cells, non-treated and treated with 0.5 µg/mL of human IgG1 isotype control or atezolizumab, were cultured for five days in a black 96-well tissue culture-treated plate (3000 cells per well). Then BrdU assay was performed according to the manufacture’s protocol. The quantity of BrdU incorporation into cellular DNA was determined by a plate-based luminometer to measure the relative light units (RLU) at 425 nM (Promega Corporation, Madison, WI, USA). Results are expressed as a fold of change in BrdU incorporation.

Cell growth assay was carried out using BrdU Cell Proliferation Chemiluminescent Assay Kit (Cell Signaling) in 96‐well plate. Cells were incubated with BrdU for 4 h. A wavelength of 430 nm was measured using a Multiskan^™ FC Microplate Photometer (Thermo Scientific).