Brdu monoclonal antibody

Cell viability was analyzed by CCK-8 assay. Briefly, CHON-001 and C28/I2 cells (3000 cells/well) were seeded into 96-well plates in 100 µL of DMEM medium. After adhering to the plates, cells were transfected with indicated vectors. 48 h after transfection, cells were stimulated with 10 ng/mL IL-1β for 24 h, followed by incubation with CCK-8 reagent (each well adding 10 µL CCK-8; Beyotime, Shanghai, China) for another 1 h. Then, the optical density value was evaluated by a Multiskan microplate reader (Thermo Fisher) at 450 nm.
The proliferation of CHON-001 cells was also evaluated by 5-bromo-12’-deoxyuridine (BrdU). After stimulation with 10 ng/mL IL-1β for 24 h, cells grown on coverslips were incubated with 20 μM BrdU solution for 2 h, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 1 mol/L HCl for 30 min. After blocking with 3% bovine serum albumin at room temperature for 1 h, cells were treated with BrdU monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4 °C. Then, cells were incubated with Alexa FluorR® 594-labeled secondary antibody (Thermo Fisher). Finally, the nucleus was stained with DAPI. A fluorescence microscope was used to capture photos. BrdU positive cell number was calculated in 5 random fields per sample.