Measuring Anti-inflammatory Effects of Compounds

The C2C12 myoblast cells were initially tested for their ability to produce pro-inflammatory cytokines (TNF-α, IL-6, nitric oxide, and IL-1β) by stimulating the cells with 100 ng/mL of lipopolysaccharide (LPS) (from Escherichia coli O111:B4 strain (L2630)) for 24 h. To observe IL-1β production, the LPS-stimulated cells were further activated by ATP (5 mM) for 30 min. The concentration of pro-inflammatory cytokines from the cell supernatant was assayed by a commercially available ELISA kit (Invitrogen mouse ELISA Kit, Life Technologies Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. The level of NO in the culture supernatant was determined using the Griess reaction. The procedure for Griess reaction has been described in detail in our previous paper by Hua et al. [35 (link)]. To study the ability of IC and AC to suppress IL-6 production, the C2C12 myoblasts and myotubes (8th-day post differentiation) were cultured in 6-well plates for 24 h and treated with different concentrations (25 µg/mL, 12.5 µg/mL, and 6.25 µg/mL) of IC and AC for 30 min and 3 h. Later, they were stimulated with 100 ng/mL of LPS for 24 h. The concentration of IL-6 from the cell supernatant was assayed by a commercially available ELISA kit (Invitrogen mouse-IL-6 ELISA Kit, Carlsbad, CA, USA) according to the manufacturer’s instructions.