Cell proliferation and drug synergy assay

To determine the effects of compounds on cell proliferation, cells were plated in 96-well plates at a density of 5000–10,000 cells per well to achieve 70% of confluence in a volume of 100 µL, and grown for 24 hr prior to treatment. Cells were then treated with DMSO or compounds at concentrations ranging from 1.5 nM to 10 µM (threefold dilutions). After 6 days, cell proliferation was measured using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) and Victor4 plate reader (Perkin Elmer, Waltham, MA). Percent inhibition was calculated relative to DMSO signal, and all results shown are the mean of triplicate measurements.
For synergy analyses of drug combinations, 5000–10,000 cells suspended in 100 µl media were plated in triplicate into each well of 96-well tissue culture plates and grown for 24 hr prior to treatments. Ceritinib and CGM097 at different concentrations defined by a dose matrix were added to the cells such that all pair-wise combinations as well as the single agents were represented. Cells were incubated for 72 hr following compound addition, and cell viability was measured using the CellTiter-Glo luminescent cell viability assay (Promega) and Victor4 plate reader (Perkin Elmer). Isobolograms and combination indices were determined as described by Lehar et al. (Lehár et al., 2009 (link)).