Type 1 collagen coated plates

Previously isolated human skeletal muscle cells (HSkMCs) from severely obese, insulin‐resistant women (BMI ≥40 kg/m², HOMA‐IR ≥2.5, n = 6) and lean insulin‐sensitive women (BMI < 25 kg/m², HOMA‐IR < 2.5, n = 6) were used in this study (Kugler et al., 2020). In brief, human skeletal muscle cells (Passage 3) were thawed, pooled together, and grown in a humidified environment with 5% CO₂ at 37°C on a type‐I collagen‐coated flask (Greiner Bio‐one). At a confluence of ~80%–90%, myoblasts were subcultured onto type I collagen‐coated plates (Corning), 35 mm collagen‐coated glass‐bottom dish (MatTek), Seahorse XFp cell culture miniplate (Agilent Technologies), or 96 well clear bottom black polystyrene microplate (Corning) depending on experimental purposes. Upon reaching ~80%–90% confluency, myoblasts were switched to low‐serum (2% horse serum) media to induce differentiation into myotubes. On day 6 of differentiation, myotubes were treated with either Mdivi‐1 (20 μM) or vehicle (1% DMSO in PBS) for 12 h. For insulin signaling and glucose uptake measurements, myotubes were serum‐starved for 3 h, incubated with or without 100 nM of insulin for 10 min, and cell lysates were collected for further analysis as previously described (Bikman et al., 2010). All experimental procedures were repeated in three independent experiments.

Primary mouse hepatocytes were isolated as described previously [12 ,13 (link)], seeded at a density of 8 × 10⁴ cells/cm² on type I collagen-coated plates (Corning Inc., NY, USA) in WME containing 10% fetal bovine serum, 2 mM L-alanyl-l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin, then cultured at 37°C in a humidified atmosphere with 5% CO₂. Forty-eight hours after seeding, cells were cultured in serum-free WME for 24 h, then used for subsequent assays. Neonatal rat cardiomyocytes were isolated from 2-day-old Sprague Dawley rat pups as previously described [14 (link)], then used for subsequent assays.