Realtime glo assay

Cell viability was assayed in situ once weekly starting the day after seeding using the RealTime-Glo assay (Promega) according to the manufacturers protocol. Briefly, MT Cell Viability Substrate and NanoLuc Enzyme were diluted 1:500 in media and 25 μl was added to each well (1/5 total final volume). Cells were incubated for 1 hour at 37°C and luminescence measured. For experiments including RFP-labeled cells, RFP fluorescence was measured using Ex 553 nm/Em 574 nm. We did not observe any effect of the presence of RFP on RealTime-Glo luminescence or vice versa. Fresh media containing gefitinib was replaced immediately after each assay.

Cell lines were seeded into 96 well plates 24 hours before addition of drug. For dose response experiments, drugs were added to cells and cell proliferation determined by CellTiter-Glo assay (Promega) after 72 hours. For experiments on DV-90 PSR resistant clones, which proliferate more slowly than parental cells, cells were incubated for 5 days to allow for the same number of cell doublings of vehicle treated cells. For long-term viability assays, cell number was determined 24 hours after seeding and prior to drug addition, and at indicated time points by RealTime-Glo assay (Promega).

For cell proliferation dose response assays, cell lines were seeded into 96-well plates 24 hours before addition of drug. Cell proliferation was determined by CellTiter-Glo assay (Promega) 72 hours after adding drug according to the manufacturers protocol. For short-term time course experiments, plates were drugged in identical fashion at indicated time points; all plates in an experiment were developed with CellTiter-Glo simultaneously. Cell viability studies were performed using crystal violet staining. Cells were fixed with glutaraldehyde, washed with water and stained with 0.1% crystal violet for 30 minutes. After imaging, staining was quantified by using 10% acetic acid to extract the stain and absorbance read at 590nm. Long-term cell viability studies were performed using RealTime-Glo assay (Promega) according to the manufacturer’s protocol. Cell viability was assessed at the indicated time points and fresh media was replaced immediately afterward. For navitoclax pre-treatment studies, we observed similar sensitization over a range of pre-treatment and washout time periods (48-72 hours); for consistency, 48 hour pre-treatment/48 hour washout was used unless otherwise indicated.