Cytotox non radioactive cytotoxic assay kit

Mesothelial cells were cultured for 48 h in 96-well plates. Complete DMEM low glucose was used as the culture medium, and the cells were treated with or without escalating concentrations of halofuginone (10, 20 or 40 nM) or tryptophanol (125, 250, 500 nM). Cytotoxicity was assessed by the lactate dehydrogenase (LDH) release assay using the Cytotox Non-Radioactive Cytotoxic Assay kit (Promega Corporation, Madison, WI, USA), and calculated by the equation Cytotoxicity (%) = (LDH in the supernatant: Total LDH) × 100.

Since the concentrations of AOAA vary in different studies, we did preliminary experiments to determine a non-toxic AOAA concentration for RPTECs. For this purpose, hamster and mouse RPTECs were cultured for 26 hours in 96-well plates in a humidified atmosphere containing 5% CO₂ in the presence or not of escalated concentrations of AOAA (0.5, 1, and 2 mM). Cytotoxicity was assessed with LDH release assay using the Cytotox Non-Radioactive Cytotoxic Assay kit (Promega Corporation, Madison, WI, USA). Cell death was calculated by the equation Cell death (%) = (LDH in the supernatant: Total LDH) × 100. These experiments were performed in triplicates and repeated nine times. As depicted in Figure 1, AOAA was not cytotoxic at concentrations of 0.5, 1, or 2 mM. The latter concentration of 2 mM was selected for all subsequent experiment.