Mtt assay protocol

Cell proliferation was determined by a colorimetric assay based on the ability of viable cells to reduce the tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (American Type Culture Collection, Manassas, VA, USA; ATCC 30-1010K) to formazan. HPAECs were grown in 96-well microplates overnight at a density of 5 × 10⁴ cells per well in 100 µL of complete medium, followed by an additional period of growth for 24 h in basal medium containing 0.5% fetal bovine serum (FBS). The cells were then treated with varying concentrations of PD98059 and grown under reduced serum conditions for up to 48 h, after which cell proliferation was assessed by the MTT reduction assay, as outlined in the MTT Assay protocol (American Type Culture Collection, Manassas, VA, USA). Briefly, at the end of the experiments, 10 µL of MTT reagent was added to each well, and the cells were incubated in a humidifier containing 5% CO₂ at 37 °C for 2 h, at the end of which precipitates were visible in all wells. Following the incubation, 100 µL of detergent was added to each well, and the cells were incubated at room temperature in the dark for additional 2 h; the absorbance was measured at 570 nm. The absorbance readings are directly proportional to the number of cells.

Cell viability was determined by a colorimetric assay based on the ability of viable cells to reduce the tetrazolium salt, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), to formazan. HPMEC were seeded onto 96-well microplates, treated with DMSO or OM, followed by exposure to air or hyperoxia for up to 48 h. The cell viability was then assessed by MTT reduction assays as outlined in the MTT Assay protocol (American Type Culture Collection, Manassas, VA).