Arrayscan vti hcs microscope

The test compounds were added to the cells 1 h after seeding for an exposure time of 24 h. The cells were stained with 1 µg/ml Hoechst (H-33342) and 1 µM calcein-AM 1 h before readout. Imaging was performed automatically using an ArrayScan VTI HCS microscope (Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed for neurite area and cell viability by an automated algorithm exactly as described previously (Hoelting et al. 2016 (link); Stiegler et al. 2011 (link)). In brief, H-33342 staining was used to identify individual cell nuclei. The somatic area was defined by enlarging the nuclear area, and subtracted from the calcein stain. The remaining calcein-positive pixels corresponded to the neurite area. Cell viability was assessed using the same images. Cells that were double-positive for H-33342 and calcein were classified viable, cells only positive for H-33342 as dead.

Immature peripheral neurons were thawed and seeded at a density of 100,000 cells/cm². For initial toxicity assessment, cells were left to attach for 1 h at 37 °C and 5% CO₂ followed by treatment with the test compounds. Cells were exposed to the compounds for 24 h, 48 h, and 72 h and readout was performed on DoD1, 2, and 3, respectively.
For delayed toxicity assessment, cells were cultured until DoD4. On DoD4, test compounds were added to the cells together by performing a half-medium exchange. Readout was performed after 72 h (on DoD7).
For the readout, neurons were stained with 1 µg/mL HOECHST-33342 (H-33342) and 1 µM calcein-AM (both from Merck, Darmstadt, Germany) 1 h prior to the imaging. After incubation for 1 h at 37 °C and 5% CO₂, images were acquired automatically using an ArrayScan VTI HCS microscope (Thermo Fisher Scientific, Waltham, MA, USA). Images were analyzed for neurite area and cell viability as previously described [86 (link)].

Cells were seeded into U-bottom 96-well plates coated with 20 mg/mL poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich) at the density of 1500 cells per well and centrifuged at 400 g for 5 min. Three-day pre-formed spheroids were treated with the different drugs at indicated concentrations during 48 h. After that, Hoechst (200 ng/mL) and PI (0.5 μg/mL) were added as markers of cell nuclei and cell death, respectively. Images of PI (BGRFR_549_15 filter) and Hoechst (BGRFR_386_23 filter) fluorescence were acquired with a 5× objective on the ArrayScanVTI HCS microscope (Thermo Fisher Scientific, Villebon sur Yvette, France) at the CMBA platform. The HCS Studio Morphology Explorer bio-application was used to automatically perform High Content image Analysis and extract parameters such as spheroid area measurement, fluorescent intensity measurement of each staining and count of the dead cells number.