Cd83 fitc

The cells were phenotypically characterized by flow cytometry using the following conjugated antibodies (Abs): mouse anti human-HLA-ABC-FITC, CD80-FITC, CD83-FITC, CD86-FITC, CCR7-FITC, and CD11c-PE-Cy7 (eBioscience, San Diego, CA, USA). Briefly, cells were gently removed from the culture plates using cell scrapers. Then, the cells were centrifuged at 1000 rpm for 5 min at 4 °C, washed with PBS and incubated with Abs for 30 min. After being washed twice with PBS, samples were acquired on a FACSVerse (BD Biosciences, Hershey, PA, USA) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Cell viability was verified through the viability dye LIVE/DEAD (Thermo Fisher Scientific Inc., Waltham, MA, USA). All the analyses were made in the CD11c+ cell population of each condition and sample.

Fc-gamma receptors were blocked with a rat anti-mouse CD16/CD32 antibody (BD). Mouse-specific antibodies were the following: CD3-Brilliant violet 510, CD25-PercP Cy5.5, I-A/I-E-Brilliant violet 510, CD45R/B220-PE-Cy7, CD43-APC, CTLA-4-APC (all from BioLegend), CD4-APC-Cy7, CD69-PE, CD11c-PercP Cy5.5, CD19-APC-Cy7, CD40-FITC (all from BD Pharmingen), CD8α-PE-Cy7, FoxP3-FITC, CD86-APC, CD83-FITC, CD80-PE-Cy7, IgM-PercP-eFluor 710 (all from eBioscience).
During the experimental set-up, CD3 antibody was included in the staining panel (BV510 BioLegend clone 17A2) and used for the gating of T cells. The results obtained with gating on CD3+ CD4+ CD8- were similar to those obtained with gating on CD4+ CD8-. Due to the limitation in the maximum number of colours that can be discriminated with the FACS Canto II, CD3 staining was omitted in subsequent stainings.
Dead cells were excluded using the fixable viability dye-eFluor 450 (eBioscience), and intracellular staining was performed using the fixation/ permeabilization buffer set from eBioscience. Flow cytometry measurements were performed on a FACS Canto II instrument (BD) and the data were analyzed with FlowJo software (Tree Star).

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.