Mouse il 1β elisa kit

IL-1β levels in the supernatant of C2C12 cells were determined with a mouse IL-1β ELISA kit (BioLegend, CA, USA) following the manufacturer's recommendations.

CellXVivo Mouse Th17 Cell Differentiation Kit (R&D Systems, Minneapolis, MN, USA) was used to induce differentiation of Th17 cells from the preparation of CD4+ T cells isolated from mouse splenocytes, according to the manufacturer’s instruction. CD4+ T cells were differentiated for a total of 5 days under Th17 polarization conditions. To assess effects of MSCs on Th17 differentiation, CD4+ T cells were cocultured with control MSCs or SAA MSCs in 24-well dishes without physical separation after 48 hours of differentiation induction. The ratio of CD4+ T cells to MSCs was 100:1. In the coculture system, MSCs could interact with CD4+ T cells through both direct cell-to-cell contact and paracrine mechanisms. For baseline control, CD4+ T cells in the basic group were differentiated for 5 days under the same conditions but without coculturing with MSCs. At the end of differentiation on day 5, the cell culture supernatant was collected and cytokine secretion was determined using the Mouse IL-17A ELISA kit (BioLegend, San Diego, CA, USA) and the Mouse IL-1β ELISA kit (BioLegend, San Diego, CA, USA). The levels were determined in triplicate.

Snap frozen lungs were homogenized in cold lysis buffer containing 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 10% Glycerol, 5 mM EDTA and a protease inhibitor cocktail. Tissue debris was pelleted by centrifugation at 4°C for 10 min at 300×g and protein concentration in the supernatants was measured at 660 nm with protein assay reagent (Thermo Fisher Scientific). Mouse osteopontin ELISA kit (RayBiotech) and mouse IL-1β ELISA kit (BioLegend) were used per manufacturer’s instructions.