Clariostar monochrome microplate reader

The CellTiter 96 Aqueous One Solution (MTS) Cell Proliferation Assay (Promega, Australia) was used to determine cell viability of C2C12 myotubes, according to the manufacturer's instructions. Briefly, C2C12 myotubes stimulated with CSE or H₂O₂ were washed and incubated in media containing MTS reagent at 37°C and 5% CO₂ for 1 h. Absorbance was then recorded at 490 nm using a plate reader (CLARIOstar Monochrome Microplate Reader; BMG Labtech, Australia). To validate the specificity of the assay, MTS reagent was added to unseeded culture plate containing CSE or H₂O₂ for 1 h before absorbance reading. No significant absorbance changes were observed in response to these stimuli verifying the reliability and specificity of the assay.

Mature IL‐6 and IGF‐1 released by the C2C12 myotubes were quantified using commercially available enzyme‐linked immunosorbent assay (ELISA) kits: murine IL‐6 ELISA Kit and murine IGF‐1 DuoSet ELISA Kit, according to the manufacturer's instructions. Briefly, plates were pre‐coated with capture antibody and then blocked with a universal diluent. Antibody standards were serially diluted in the universal diluent, constructing a 7‐point curve with a universal buffer as blank. Cell supernatant (undiluted) was then added in duplicates into the appropriate wells and agitated on a Thermomixer (Eppendorf, Germany) at 800 rpm for ≥2 h at room temperature. Wells were thoroughly washed with 0.05% Tween 20 in 1× PBS (PBST) before the detection antibody was added and agitated for 1 h at 800 rpm at room temperature. After washing, a developing solution with reporter enzyme and substrate was added and agitated for a further 1 h at room temperature. Absorbance was then recorded at 450 nm using a plate reader (CLARIOstar Monochrome Microplate Reader; BMG Labtech, Australia).