Luminex based multiplex assays

Levels of IL-6, CCL2, CXCL1, and TNFα were quantified using DuoSet ELISA kits according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). Absorbance was read at 570 nm and background was removed using 450 nm absorbance on a Synergy HTX plate reader and Gen5 software (BioTek). For measurements on gastrocnemius muscle, tissue (>100 mg) was placed within gentleMACS M Tubes (Miltenyi Biotec, Waltham, MA) containing 500 μL PBS and protease inhibitor cocktail (MilliporeSigma) and lysed on a gentleMACS Dissociator according to manufacturer’s settings. Lysate was then passed through QIAshredder spin columns (Qiagen, Germantown, MD, USA). Total supernatant protein content was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). In some experiments, plasma and cell culture supernatants were analyzed by a custom Luminex-based multiplex assays (R&D Systems) for mouse IL-6, TNFα, CCL2, CXCL1, and IL-1β, according to manufacturer instructions. In some instances, data were extrapolated beyond the upper and lower limits of quantitation by the BioPlex Manager Software (Bio-Rad) and used for graphical presentation and statistical analyses.