Mouse il 17a elisa kit

Fluorochrome-conjugated monoclonal antibodies (mAbs), including PE-CD11b (M1/70, 101207, 1:400), PerCP/Cy5.5-IL-17A (TC11-18H10.1, 506919, 1:100), PE/Cy5-Gr-1 (RB6-8C5, 108409, 1:600), APC-CD4 (GK1.5, 100412, 1:400), FITC-TNF-α (MP6-XT22, 506304, 1:100), and APC/Fire750-CD4 (GK1.5, 100460, 1:100) were purchased from BioLegend (San Diego, CA), while PE-IL-17A (eBio17B7, 12-7177-81, 1:100) was purchased from eBioscience (San Diego, CA). Anti-mouse IL-17A neutralizing monoclonal antibody (17F3, BP0173) was purchased from BioXCell (West Lebanon, NH). Recombinant mouse IL-1β, IL-6, IL-17A, and TNF-α were purchased from BioLegend. Mouse IL-17A ELISA kit was purchased from BioLegend. Mouse IL-17A ELISPOT kit was purchased from R & D Systems (Minneapolis, MN). Native chicken and bovine collagen type II were purchased from Sigma-Aldrich (St. Louis, MO) and Chondrex (Redmond, WA), respectively.

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.