Ctg luminescent cell viability assay

Human cancer cell lines cultured in the complete growth medium were seeded into 96-well plates containing DMSO or test compounds. The seeding density for each cell line (typically 2000–3000 cells per well) was optimized to allow the cell growth in the linear range during culture period (5–7 days). After the culture period, cell proliferation was assessed using CTG Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Individual compound test AUC was calculated with 10-point CTG assay data (0–10 µM, half-logarithmic dilution).

Ex vivo cultures were prepared as described previously^{38 (link)}. Briefly, patient tumor tissues were mechanically disaggregated and treated for 1 h with 125 U/mL collagenase and 2.5 mg/mL DNAse (both from Sigma-Aldrich). To remove aggregates and debris the cell suspensions were filtered through 100 μM nylon Cell Strainer (BD Falcon, Franklin Lakes, NJ) and washed with ice-cold PBS. Red blood cells were removed using ACK lysing buffer (Lonza, Basel, Switzerland). Live cells were seeded out at density of 15–20 × 10⁵ cells/well in Nunc™ 96-Well Polystyrene Round Bottom Microwell plates (Thermo Fisher Scientific, Waltham, MA) in RPMI++ medium supplemented with 100 units/mL penicillin and 0.1 mg/mL streptomycin (both from Lonza) and allowed to form 3D spheroids. Drugs were immediately added to the cells and left for five days before viability was assessed using the CTG Luminescent Cell Viability Assay (Promega) and analyzed by Fluoroscan Ascent Fl (Thermo Fisher Scientific). The ex-vivo assay was performed once for each patient sample, with at least three technical replicates per condition.