High-Throughput Viability Screening of Drugs

Each drug was assayed at six concentrations. Each cell line was seeded on two 384-well plates with 4,000 cells per well and four replicates per plate. Each plate included a control for background fluorescence signal, 10% dimethyl sulfoxide (DMSO), and another control for drug vehicle, either water or DMSO at 0.1%, with the exception of temozolomide, which had DMSO at 0.08%, 0.20%, 0.41%, 0.62%, 0.82%, and 1.23%. Each cell line was incubated for 72 h with each of the treatments for all tested concentrations, dyed with alamarBlue (BioSource International), and incubated for another 24 h. The alamarBlue assay is a fluorometric and colorimetric cell viability assay incorporating an oxidation-reduction indicator that responds to cellular metabolic reduction, with the intensity of fluorescence produced proportional to the number of living cells. Subsequently, a Tecan Freedom EVO 150 robotics system with a Connect stacker and F200 plate reader, which measures fluorescence intensity in raw fluorescence units (RFUs), was used for viability measurements. We applied quality control procedures and calculated cell viability as previously described [1 (link), 22 , 29 (link)].