Cellxvivo mouse th17 cell differentiation kit

Naïve CD4⁺ T cells (>99% purity, CD62L^hiCD44⁻) were isolated from spleens of WT or Nod2^−/− mice by negative selection (StemCell). Cells were then differentiated in vitro according to manufacturer’s instruction (CellXVivo mouse Th17 cell differentiation kit, R&D systems): 1.25 × 10⁵ naïve CD4⁺ T cells were incubated in serum-free X-Vivo 15 medium (Lonza) containing penicillin/streptomycin and in the presence of CD3 mAb, CD28 mAb and Th17-polarizing cytokines as provided by the kit. After 5 days of culture, CD4⁺ T cells were stimulated with PMA/ionomycin in the presence of brefeldin A and the frequency of IL-17⁺ CD4⁺ T cells was quantified by flow cytometry (as above).

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.