Apc conjugated anti il 17 mab

For intracellular cytokine staining, cells were incubated for 3 h with 100 ng/mL of PMA and 1 μM of ionomycin (all from Sigma-Aldrich, Saint Louis, MO, USA), brefeldin A, and monensin (all from eBioscience, San Diego, CA, USA). After washing cells with cold PBS containing 1.5% FBS, cells were stained with APC-Cy7-conjugated anti-CD45.2 mAb and PE/Cy7-conjugated anti-CD4 mAb (eBioscience, San Diego, CA, USA) for surface staining. Cells were then washed and stained with PerCp-Cy5.5-conjugated anti-IFNγ mAb, APC-conjugated anti-IL-17 mAb (all from BioLegend, San Diego, CA, USA) and PE-conjugated anti-RORγt mAb (eBioscience, San Diego, CA, USA) after incubation with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for 30 min at 4 °C. Cells were analyzed by LSR III flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analyzed with FlowJo (TreeStar, Ashland, OR, USA).