Pe cy7 anti ly6c

Splenocytes were stained with FITC-anti-Gr-1, PE-anti-CD11b PE-Cy7-anti-CD11c, PerCP-Cy5.5-anti-CD3 (BD Pharmingen, USA) and APC-anti-Ly6G, PE-Cy7-anti-Ly6C, APC-anti-F4/80, and APC-anti-I-A^b (Biolegend, USA) antibodies. For intracellular cytokines staining, DLN cells and PBMC were stimulated with 50 ng/ml phorbolmyristate acetate (PMA) plus 500 ng/ml ionomycin (both from Sigma, USA) for 5 h in the presence of 10 μg/ml brefeldin A (eBiosciences, USA). Cells were fixed, permeabilized, and stained with PE-anti-IL-17A (BD Pharmingen, USA) after staining with FITC-anti-CD4 antibodies. To analyze the frequency of Foxp3⁺regulatory T cells (Treg) in DLN, after stained with FITC-anti-CD4 and PE-anti-CD25 (eBioscience, USA) antibodies, cells were fixed, permeabilized, and stained with PE-Cy5-anti-Foxp3 antibody (eBioscience, USA). Data were acquired using Beckman flow cytometer (USA).