Il 17 percp cy5

CD4⁺ T cells were purified from lymph nodes and spleens using Mouse CD4⁺ T Cell Isolation Kit (eBioscience, San Diego, CA, USA) and activated with plate-bound anti-CD3 (eBioscience, clone:145-2C11, 1 μg/mL) and anti-CD28 (eBioscience, clone: 37.51, 2 μg/mL) antibodies for indicated times. T cell proliferation was assessed by Ki67 intranuclear staining following fixation and permeabilization in the Foxp3/Transcription Factor staining kit (eBioscience). For intracellular staining of cytokines, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 500 ng/mL, Sigma Chemical Co., St. Louis, MO, USA) and ionomycin (1 μg/mL, Calbiochem Corp., La Jolla, CA, USA) for 5 h at 37 °C in the presence of Brefeldin A (10 μg/mL, eBioscience). Cells were stained with anti-CD4-FITC antibody (eBioscience), fixed, permeabilized and then stained with anti-IFNγ-PE, anti-IL-4-PE, IL-17-PerCP-Cy5.5, Tbet-PE, or RORγt-PE antibody, as per the manufacturer’s instructions (eBioscience). Sample acquisition was performed with Beckman Coulter CytoFlex (Beckman Coulter, Brea, CA, USA) and data were analyzed using CytExpert 2 software (Beckman Coulter, Brea, CA, USA). Plots were prepared using CytExpert 2 and FlowJo (San Carlos, CA, USA) software.