Cellomics arrayscan platform

Proliferation of M21 cells was measured using the Click-iT EdU Alexa 647 HCS assay (Thermo Fisher Scientific) following manufacturer's instructions. 5000 cells per well were seeded in a black, clear bottom 96 well plate. Six wells per cell line were seeded, cultivated overnight and then incubated with 10 μM EdU for 3 hours under standard cell culture conditions. Detection and quantification of EdU positive cells was performed using an Cellomics Arrayscan platform (Thermo Fischer Scientific). At least 1700 cells per well were detected by DAPI staining and EdU incorporation analyzed.

Cell viability was assessed using a three-color fluorescence assay and high-content imaging. For DOP treatment and siRNA-mediated knockdown, 6000 and 2500 striatal cells, respectively, were seeded in sterile, black 96-well microplates. For DOP treatment, cells were incubated for 24 h in phenol red-free and serum-free DMEM, containing either vehicle (DPBS) or 4 mM DOP. For knockdown of target genes, cells were transfected for 48 h with the appropriate siRNAs. siRNA transfected cells were washed with 1× DPBS and further incubated for 24 h in serum-free DMEM. After the 24-h incubation, Calcein AM (Thermo Scientific, C3099—final concentration: 1 µg/ml), Propidium Iodide (Thermo Scientific, P3566—final concentration: 2 µg/ml) and Hoechst 333442 (Thermo Scientific, H3570—final concentration: 2 µg/ml) were added to quantify live, dead, and total cells, respectively. After 20 min incubation at 33 °C, image acquisition was carried out with a Cellomics Arrayscan Platform (Thermo Scientific). Seven fields per well were scanned at 10× magnification and quantitative analysis was performed using Cellomics proprietary algorithm for cell viability. Cell loss was calculated as the ratio of Propidium Iodide-positive cells to the total cell counts.