Cellinsight cx7 microscope

Cell cultures were fixed (10 min, 4% PFA), permeabilized (10 min at −20 °C with ice cold MeOH), blocked (1 h, 5% BSA) and stained using antibodies against USP18, ISG15 and MyHC (Supplementary Table S2). USP18 antibody was incubated overnight at 4 °C whereas all other antibodies were incubated for 1 h at room temperature. Muscle bundles were stained for titin as previously described [60 (link)]. Imaging of both 2D and 3D muscle models were carried out using the CellInsight CX7 microscope and a cell-based analysis was carried out using the accompanied HCS Platform software (Thermo Fisher Scientific). 3D muscle bundles were imaged with 20X objective using confocal for 40 stacks at 5 µm, images show maximum projections. Fusion index indicates the percentage of segmented nuclei within MyHC positive object, the myotubes, compared to the total number of nuclei.

We employed HCA to quantify the neuronal branching outgrowth and complexity using CellInsight CX7 microscope (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, we split NPCs or DNs at 4 or 8 weeks of differentiation using Accutase and seeded them at a density of 10,000 cells/well on Matrigel-coated 96-well plates with black-wall and clear-bottom (Corning, Corning, NY, USA, #353219). We then stained the cells with TUJ1 antibody (Sigma-Aldrich, St. Louis, MO, USA, #T8578; 1:3000) using 4% PFA (EMS, Thermo Fisher Scientific, Waltham, MA, USA, #50980487) for 20 min at RT and washed two times with PBS. For permeabilization, we incubated the fixed cells with a blocking solution containing 10% normal donkey serum (DNS) and 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA, #T8787) in PBS with 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA, #P9416) (PBS-T) for 1 h at RT. We diluted primary antibodies in blocking solution, incubated them overnight at 4 °C on a shaker, and performed counterstaining with Hoechst. The morphogenesis of TUJ1-positive cells within NPCs or DN cultures were quantified using the “Cellomics Neuronal Profiling v4 BioApplication” (CellInsight CX7, High Content Platform, Thermo Fisher Scientific, Waltham, MA, USA). We used a similar HCA-based approach to quantify the number of TUJ1-positive neurons and TH-positive neurons within DNs.

For the proof-of-concept compound screening, 48 compounds were selected from a library of 700 FDA-approved drugs (Selleckchem- z65122) and 5 drugs were added, used in the context of AUD treatment (Appendix Table 1). Briefly, 10,000–20,000 DNs/well were plated on black-wall, round bottom 96-well plates (μClear, Greiner) 5 days before adding the compounds ON. Compounds were used in a final concentration of 0.5 μM diluted in culture media. DMSO concentration was diluted down below 0.05%. The second day, DNs were stained with 0.5 nM TMRM together with 1:10,000 Hoechst diluted in phenol red-free culture medium for 30 min at 37°C, 5% CO₂ by adding the staining solution into the media containing the drugs. After removal of the staining solution, compounds together with 1 M ethanol were applied to the DNs and incubated for 1 h at 37°C, 5% CO₂. Control wells included DNs kept untreated and DNs exposed to 1 M ethanol only. HCI was conducted with the CellInsight CX7 microscope (Thermo Fisher) and analyzed according to the “CellHealth Profiling” and “Neuronal Profiling” BioApplication. The same screening was repeated twice, and the values shown in Figures 6B–D represent the mean values of the two runs (mean, ±SD).