Evos fl auto 2 inverted

Myotube area was measured as previously described.²³, ²⁴ Treated myotubes were gently washed with PBS, fixed with 1:1 methanol:acetone for 10 min at −20°C, left to air dry for 10 min, and incubated with primary antibody against sarcomeric myosin (MF 20 was deposited to the DSHB by Fischman, D.A.) (Product MF 20; DSHB Hybridoma Bank, USA) at 1:25 in blocking solution (PBS + 5% goat serum + 1% BSA) for 1 h at 37°C. Cells were then washed 3× in PBS, followed by incubation with 1:250 secondary antibody (AlexaFluor594, mouse IgG2b; ThermoFisher, USA) for 1 h at 37°C. Cells were imaged using automated capture routines on an Evos FL Auto 2 inverted fluorescent microscope (ThermoFisher, USA) and analysed using custom written routines in CellProfiler (Broad Institute, USA) to assess MF 20+ area (myotube area). All processing procedures were performed uniformly over the entire set of images using batch processing modes to avoid any human bias.