Fluostar optima automated plate reader

The drug screen and DSS calculation were conducted as previously described [23 (link)]. Drugs with a DSS score > 10 were considered effective. The library [24 (link)] was dispensed on 384-well plates by the High Throughput Biomedicine core unit (Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, Finland), each drug in five different concentrations (in technical duplicates) on a 10,000-fold concentration range. Cells were seeded at a density of 500 cells/well. For the combination screen, a cell line specific EC25 of entinostat (Suppl. Table 1, Suppl. Figure 1A) was added using a Tecan D300e Digital Dispenser (Tecan Life Sciences, Männedorf, Schweiz). After 72h cell viability was measured with CellTiter-Glo®2.0 Cell Viability Assay (Promega, G9243) (CTG) on FLUOstar OPTIMA automated plate reader (BMG Labtech).

Measurement of metabolic activity in DKFZ-BT66 cells was performed in 96-well flat bottom black opaque wall plates (Greiner) after inhibitor treatment for 72 or 240 hours. 24 hours before treatment 25.000 cells/well were seeded for experiments with 72 hours duration and 5.000 cells/well were seeded for experiments with 240 hours duration in experiments in the presence of 1μg/ml doxycycline. For experiments in the absence of doxycycline 100.000 cells/well were seeded 24 hours prior to treatment for both, the short and the long-term treatment time. Cells were incubated with the different MAPK inhibitors in several dilutions, ranging from 10 μM to 0.005 nM. All treatments were performed in triplicate. Each plate contained an untreated and a DMSO control. Medium was exchanged three times per week and replaced in order to keep inhibitor levels and doxycycline levels stable. After the incubation time metabolic activity was determined using CellTiter-Glo assay (Promega, catalogue no. G7571) following manufacturer´s instructions. Luminescence was detected by FLUOstar OPTIMA automated plate reader (BMG Labtech). Dose-response curves and half maximal inhibitory concentrations (IC₅₀-values) were calculated using GraphPad Prism software (v5.01) for windows.