Cd1a fitc

The following antibodies were used for flow cytometry analysis: CD11c APC, CD80 FITC, CD83 FITC, CD86 PE, HLA-DR FITC, CD1a FITC, CD14 FITC, CD4PE, CD4 PE-Cy7, CD45RA PE, CD45RO FITC, CD25 PE-Cy7, CTLA-4 APC, CD39 PerCP-eFluor710, FOXP3 Alexa Fluor 700, IL-10 PE, IFN-γ PE-Cy7, and IL-17 APC (all eBioscience). For surface staining, cells were incubated in PBS 10% FBS containing the respective antibodies for 30 min at 4°C, washed, and stored in IC fixation buffer until analysis (eBioscience). Intracellular FoxP3 was detected using FoxP3-staining kit (eBioscience) according to the manufacturer’s instructions. For intracellular cytokines detection, cells were treated with 50 ng/ml PMA, 1 μg/ml ionomycin, and 1 μl/ml brefeldin A for 5 h. After harvesting, cells were surface stained for CD4. Intracellular cytokine staining was performed in permeabilization buffer (eBioscience) before cells were washed and resuspended in FACS buffer for flow cytometry analysis. Cell viability was assessed by 7-AAD and annexin-V PE staining (eBioscience). Data were collected on FACSCalibur and FACSAria cytometers (Beckton Dickinson, San Diego, CA, USA) and analyzed with Weasel v3.0.2 software.