Labeled anti il 10 jes5 16e3

For analysis of surface markers, cells were stained in PBS containing 2% (wt/vol) BSA. Intracellular staining of the transcription factors Foxp3 was performed using the Foxp3 Fix/Perm Buffer Set (Biolegend). For detection of intracellular cytokines, cells were first stimulated for 4 h with 50ng/ml PMA and 1 µg/ml ionomycin in the presence of Brefeldin A (All obtained from Sigma), followed by staining for surface markers. Cells were then fixed and permeabilized using the Foxp3 Fix/Perm Buffer Set (Biolegend) and stained for intracellular cytokines. The following antibodies were used at a dilution of 1/200–1/600: PerCP-Cy5.5, PE-, FITC- or APC-labeled anti-IL-17 (TC11-18H10.1), PE- or APC-labeled anti-IL-10 (JES5-16E3), PE-labeled anti-Foxp3 (FJK-16s, eBioscience), PE-, FITC- or APC-labeled anti-CCR6 (29-2L17), PE-, FITC- or APC-labeled anti-CD4 (RM4-5), PE-Cy7-labeled anti-CD3 (145-2C11), APC- or PE-Cy7-labeled anti-IFN (XMG1.2), FITC-, PerCP-Cy5.5 or Pacific Blue-labeled anti-CD45 (30-F11), PE-anti-CCR4 (2G12), PE- or FITC-labeled anti-CCR9 (CW-1.2). All antibodies were obtained from Biolegend unless otherwise noted. Flow cytometry data were acquired on a 5-color FACScan (Becton Dickinson), and analyzed using FlowJo software (Treestar). Cell sorting was performed using a FACSAria II.