96 well or 6 well plates

Primary cortical neurons were separated from neonatal SD rats within 1 day of birth. The obtained small cortical tissue was placed in a culture ware containing 2 mL DMEM (high-glucose) liquid. Cortices were harvested and cut into approximately 1-mm³ small pieces, then digested with 0.05% trypsin (Gibco, Rockville, MD, United States) at 37°C for 10 min. Then, the high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) was added to the culture ware to stop digestion. The tissue suspension was centrifuged at 1000 rpm for 10 min, resuspended, and plated in 96-well or 6-well plates (Corning, Corning, NY, United States) coated with poly-d-lysine and laminin (Sigma-Aldrich, St. Louis, MO, United States) at a density of 5 × 10⁵ cells/ml with a neurobasal medium. After this, half of the neurobasal medium was refreshed every 3 days.

Mouse skeletal muscle cell line C2C12 (American Type Culture Collection, Manassas, VA, USA) was grown to confluence in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-glutamine (Gibco), 1% penicillin, and 1% streptomycin, at conditions of 37 °C and 5% CO₂. Then, cells were seeded in 96-well or 6-well plates (Corning, Corning, NY, USA) coated with 2% gelatine (Sigma-Aldrich, Saint Louis, MO, USA) and (i) kept in culture for 48 h for studies in the proliferative stage or (ii) washed with phosphate buffer saline (PBS) 1× on day 5 and then cultured in differentiation medium (DMEM supplemented with 2% horse serum, Sigma-Aldrich) for 72 h for studies in commitment stage. At these time points, cytokines IL-1β and TNF-α (BioLegend, San Diego, CA, USA) at 10 ng/mL were added. Treatments of C2C12 cells with these cytokines in the proliferation and commitment stages had no effect on cell viability (results not shown).