Cellomics arrayscan vti hcs 700 series

L4040 (15,000/well) and L5345 (15,000/well) cells were seeded in 96-well plates and allowed to attach overnight. Subsequently, cells were treated with the epigenetics compound library (Table S3) at a final concentration of 1 µM. After 72 h of treatment, cells were fixed with 4% formaldehyde and stained with 2 µg/mL Hoechst 33342 (H1399, Invitrogen Life-Technologies). An automatic nuclei count was performed with the Cellomics ArrayScan VTI HCS 700 series and HCS Studio Cell Analysis Software (ThermoFisher Scientific). Data were normalized to the negative control (i.e., 0.1% DMSO) to obtain the percent of control values. The screen was performed once in triplicate. A schematic overview of the epigenetics compound screen is depicted in Figure 2A.
The efficacy of compounds that reduced cell proliferation with >60% in both cell lines was validated by repeating the screen at final concentrations of 0.1 µM, 0.5 µM, and 1 µM. The validation screen was performed once in triplicate. A schematic overview of the validation screen is depicted in Figure 2C. The online tool MORPHEUS (Broad Institute, Cambridge, MA, USA) was used to generate heatmaps.

Chondrosarcoma cell lines were seeded in 96 well plates (3 × 10³ to 15 × 10³ cells/well) and were allowed to attach overnight. Cells were treated with different compounds (i.e., talazoparib, temozolomide, cisplatin, and doxorubicin) in concentrations ranging from 0 to 316 µM. The solvents DMSO and PBS were used as negative controls. After 72 h of treatment, cell viability was measured with PrestoBlue cell viability reagent (A13262, Invitrogen Life-Technologies) according to the manufacturer’s protocol. Fluorescence was measured at 560/590 nm with the Victor3V 1420 multilabel counter (Perkin Elmer, Groningen, The Netherlands) after 1 to 1.5 h of incubation at 37 °C. Subsequently, cells were fixed with 4% formaldehyde (Q Path, VWR Chemicals, Radnor, PA, USA) and stained with 2 µg/mL Hoechst 33342 (H1399, Invitrogen Life-Technologies). Nuclei were counted with the Cellomics ArrayScan VTI HCS 700 series and HCS Studio Cell Analysis Software (ThermoFisher Scientific, Waltham, MA, USA). To correct for growth rate, data were normalized to the “time 0 measurement” (i.e., cell viability or nuclei count before treatment) with GR Calculator (http://www.grcalculator.org) [41 (link)]. Experiments were performed in triplicate and repeated three times.