LDH cell cytotoxicity assay and AlamarBlue cell viability assay

M-huPMCs and P-huPMCs (P0) were detached from culture dishes using TrypLE enzyme (Thermo Fisher Scientific) and seeded at a density of 1 × 10⁴ cells/well in 96-well plates. Cells were cultured to 80% confluence in DMEM/10% FBS and then starved overnight with Basal Medium Eagle (Gibco) supplemented with 1% FBS, 1% penicillin-streptomycin (P/S) and 1% Insulin-Transferrin-Selenium (ITS, Thermo Fisher Scientific). M-huPMCs and P-huPMCs (P1) were then treated with 100 μM H₂O₂, 15 μM CBR-5884 and 100 μM H₂O₂ + 15 μM CBR-5884 for 6 hr, respectively. A medium only, no treatment group was included as a control. The LDH concentration in each group was assayed by LDH cytotoxicity assay kit (Pierce). In brief, 20 µl supernatant of the 96-well plate was transferred into a 386-well plate. 20 µl reaction mixture was added into each well of the plate. The mixture was incubated at room temperature for 30 min. 20 µl stop solution was added to cease the reaction. The absorbance was measured at 490 nm and 680 nm with the plate reader (Safire², Tecan). To assess cell viability, 15 µl alamarBlue reagent was added to 150 µl medium in each well of the 96-well plate. After a 2 hr incubation at 37°C, the absorbance was read at 570 and 600 nm with the Tecan Safire plate reader (TECAN).