Mammalian cytotoxicity assay

Lead compounds were tested in vitro against two mammalian cell lines to evaluate cytotoxicity. CRL-8155 human lymphocyte and HepG2 human hepatocyte cells (ATCC, Manassas, VA) were seeded in 96-well plates and incubated at 37°C for 48 h in the presence of test compound (serial-2 dilutions, in triplicate). At the end of the incubation period, cells were visually assessed before alamarBlue^TM (Thermo Fisher, Waltham, MA), a resazurin-based cell viability reagent which measures metabolic activity, was added to the plates and fluorescence measured on a BioTek FLx-800 microplate reader (BioTek Instruments, Winooski, VT). Fluorescence signals resulting from cell viability changes were compared with control wells to calculate 50% cytotoxic concentrations (CC₅₀) values.