Multiskan spectrum microplate photometer

In total, 3×10³ cells/well were seeded into a 96-well plate and cultured at 37°C for 24, 48 and 72 h. Cell proliferation was analyzed using a BrdU cell proliferation ELISA kit (cat. no. ab126556; Abcam), according to the manufacturer's protocol. Briefly, at each time point, 20 µl BrdU label was added and incubated at 37°C for 12 h. Following the incubation, the cells were fixed with 3.7% formaldehyde diluted in PBS at room temperature for 30 min and then washed three times with PBS. Then, 100 µl anti-BrdU monoclonal detector antibody (1:2,000; supplied in the BrdU cell proliferation kit) was incubated with the cells for 1 h at room temperature. Following the incubation, each well was washed three times using PBS and 100 µl horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:2,000; supplied in the BrdU cell proliferation kit) was added and incubated for 30 min at room temperature. The wells were washed three times with PBS and then, 100 µl 3,3,5′,5′-Tetramethyl benzidine peroxidase substrate was added and incubated for 30 min at room temperature in the dark. Finally, 100 µl stop solution (supplied in the BrdU cell proliferation kit) was added and the optical density value was measured every 24 h using the Multiskan™ Spectrum microplate photometer (Thermo Fisher Scientific, Inc.) at 450 nm.