The C2C12 mouse skeletal myoblasts were obtained from Pythonbio (Guangzhou, China). The cells were grown in DMEM supplemented with 10% FBS and 1% P/S. At 70–80% confluence, the cells were cultured in DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin for six days to induce differentiation into the myotubes [32 (link)]. All the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The culture media were replaced every two days.
After differentiation, the cells were seeded at 1 × 104 cells per well into black-bottom 96-well plates (Corning, Kennebunk, ME, USA) overnight for the 2D culture. The cells were seeded at 2000–3000 cells/well into ultra-low attachment 96-well round-bottomed plates (Corning, Kennebunk, ME, USA) and then cultured for three days to generate skeletal muscle spheroids (3D culture) [33 (link)]. The cells were then treated with varying concentrations of kaempferol (50, 25, 12.5, and 6.25 μM) for 24 h in the 2D culture and 48 h in the 3D culture. The treatment concentration of kaempferol was determined based on our previous cell activity experiments. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The testing was subsequently commenced.
After differentiation, the cells were seeded at 1 × 104 cells per well into black-bottom 96-well plates (Corning, Kennebunk, ME, USA) overnight for the 2D culture. The cells were seeded at 2000–3000 cells/well into ultra-low attachment 96-well round-bottomed plates (Corning, Kennebunk, ME, USA) and then cultured for three days to generate skeletal muscle spheroids (3D culture) [33 (link)]. The cells were then treated with varying concentrations of kaempferol (50, 25, 12.5, and 6.25 μM) for 24 h in the 2D culture and 48 h in the 3D culture. The treatment concentration of kaempferol was determined based on our previous cell activity experiments. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The testing was subsequently commenced.
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