Ham s f10 nutrient mixture medium

Primary myoblasts of control and Islr cKO mice were established by the method of differential adhesion. We cultured primary myoblasts in Ham’s F10 nutrient mixture medium (Gibco) containing 20% FBS and 5 ng/ml bFGF. To induce differentiation, primary myoblasts were incubated with DMEM supplemented with 2% HS.

U251 and U87 cell lines were purchased from the cell bank of the Chinese Academy of Sciences, and human astrocyte (HA) cell lines were purchased from ScienCell Laboratory. U251 and U87 cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (Beyotime) at 37°C and 5% CO₂. HA cells were cultured in Ham's F‐10 nutrient mixture medium (Gibco) containing 10% FBS and 1% penicillin and streptomycin at 37°C and 5% CO₂. For curzerene treatments, cells were treated with high glucose DMEM containing 50, 100, or 200 μM of curzerene for 12, 24, or 48 h.

Primary myoblasts were isolated from the LD muscle of pigs. In brief, the minced muscles were digested using 0.2% type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) and 2.5 U/mL dispase (Roche Applied Science, Nutley, NJ, USA) for 1 h. The filtered cell suspension was centrifuged at 1000× g for 5 min and cultured in Ham’s F10 nutrient mixture medium (Gibco, Waltham, MA USA), which was supplemented with 20% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco), and 5 ng/mL basic fibroblast growth factor (PeproTech, Cranbury, NJ, USA). The cells were incubated in an atmosphere of 5% CO₂ atmosphere at 37 °C. DMEM supplemented with 2% horse serum (HS; Gibco) was used to induce myogenic differentiation.