Multiparametric Cell Viability Assay

Unless stated otherwise, cells were seeded at 1,000 cells/well into flat-bottom 96-well plates (Greiner 655180). Edge wells were excluded and plates were left for 30 min at room temperature before transferring to an incubator. Plates for real-time analysis were transferred to the IncuCyte FLR imaging system (Essen BioScience) as previously described (10 (link)). Unless stated otherwise, compounds were added (5 μL/well) at two days post seeding, for five days. Compounds were reconstituted in dimethyl sulfoxide (DMSO). Plates were assayed for cell viability using the resazurin reduction assay and/or cell counts as described previously (10 (link)). The Spark 20M (TECAN) was used to quantify resazurin fluorescence following best practice procedures described previously (11 (link)). The ImageXpress system (Molecular Devices) was used to image Hoechst 33342 stained plates. CellProfiler (www.cellprofiler.org) was used to enumerate cell counts as previously described (12 (link)). Normalized cell viability was calculated as follows: sample/DMSO × 100. Relative half-inhibitory concentrations (EC₅₀) were calculated in Prism from the nonlinear regression algorithm of the normalized cell viability plotted against an 8-point compound dilution.