Celltiter glo 2d

Caspase-3 activity, cell viability and anoikis-recovery assays were performed using Caspase-Glo 3/7 Assay, CellTiter-Glo 2D or 3D Cell Viability Assay (Promega, Madison, WI) as described in detail in the Supplementary Methods. In the anoikis-recovery assay, cells were cultured in ultra-low attachment plates for 24 h before they were re-plated to the collagen I-coated 96-well plates to recover for 48 h.

Dissociated patient and PDX-tumour cells were seeded on 384-well plates at 2000 cells/well in 25 μL of Iscove’s Modified Dulbecco’s Media, 20% foetal calf serum and 1× insulin–transferrin–selenium (Life Technologies). Human neuroblastoma cell content in the PDX tumour was determined by flow cytometry. After 72 h, cells were treated with a library of 165 anticancer drugs. After a further 72 h, Cell-titer glo 2D (Promega) was added to measure ATP as a surrogate for cell viability. Cell viability as a function of drug concentration was plotted with a three-parameter sigmoidal dose–response curve to determine area under the curve (AUC) values. Correlation of drug sensitivity (AUC values) in patient tumour cells compared with that for PDX-tumour cells was assessed using Pearson’s correlation analysis.