Cd11c apc cy7

Flow cytometric analyses were conducted using an LSR II flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Version 7.6.5; Flowjo). All antibodies against human or mouse cells were used at appropriate dilutions, as determined by previous titration. Doublet discrimination was carried out and non-viable cells were excluded by 4,6 diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich). Mouse cells were characterized using the antibodies as follows: CD3-V500, CD4-V450, CD11c-APC Cy7, SiglecF-AF647, Ly6G-FITC, CD11b-V500, B7-1-V450, B7-2-PE Cy7, CD62L-FITC (BD Biosciences); mPDCA1-APC, B220-PE (Miltenyi Biotec); CD8-PE Cy7, F4/80-PerCP, SiglecH-PE, TLR4-AF488, TLR9-FITC, MHC-I-FITC, MHC-II-V450, PD-L1-PerCP, ICOS-L-PE, OX40L-APC (eBioscience); TLR7-PE (Abcam); p75NTR-AF488 (Advanced Targeting Systems), CD45-Pacific Blue (Biolegend), and panTrk-FITC (Cell Signaling Technology). Human cells were characterized using the antibodies as follows: TrkA-PE (R&D Systems); BDCA-2-FITC, BDCA-4-PE, p75NTR-APC, p75NTR-FITC, p75NTR-PE (Miltenyi Biotec); CD45-V500, CD3-PE, CD4-FITC, CD8-PerCP, FcεRIα-FITC, IL-3R-PE Cy7, CD184-PE Cy7, MHC-I-PE Cy7, MHC-II-PE, CD80-V450, CD86-PE, CD83-V500, OX40L-V500, PD-L1-PE Cy7, CCR7-V450, CCR9-APC (BD Biosciences), CD3-APC eFluor780, CD4-APC, CD8-PE Cy7, CD25-PE (eBioscience), and CD69-PerCP Cy5.5 (BioLegend).

For DC analysis in the skin and sdLNs, cells were stained with different combinations of the following antibodies: MHC-II-Vioblue, CD11c-PerCPVio700, CD11b-VioGreen, CD11b-PerCPVio700, XCR1-Viobright-FITC, EPCAM-PE, CD86-PE-Vio770, CD86-APC-Vio770, PD-L2-PE-Vio770 (all from Miltenyi Biotec), CD11c-APC-Cy7 (BD Biosciences), XCR1-BV510 (Biolegend). Dead cells were excluded from analysis using Zombie dye (Biolegend) of appropriate color depending of the antibodies used.
For Tregs analysis, cells were stained with combinations of the following antibodies: anti-mouse CD4-FITC (Miltenyi), CD25-APC-Cy7, CD62L-PE-Cy7 (all from BD Biosciences, Le Pont de Claix, France), Latency-associated peptide (LAP)-PE and Foxp3-APC (from e-Bioscience, Paris, France), or control isotypes. Intracellular staining was performed after fixation and permeabilization, using Foxp3 Perm Kit (e-Bioscience, Paris, France). Dead cells were excluded from analysis using Zombie dye aqua (Biolegend).
Flow cytometry was performed on a MACSquant cytometer and analyzed using FlowJo software.

Single-cell suspensions of BM and skin-infiltrating leukocytes were incubated at 4 °C for 20 min in staining buffer (1 × PBS with 0.1% bovine serum albumin and 0.1% sodium azide) containing the appropriate antibody mix. NOS2-AF488, CD45.1-eFluor 450, Ly6G-FITC, Ly6G-PE, Ly6G-PE.Cy7, CD11b-PE, CD11b-APC, F4/80-PE, CD115-APC, Ly6C-APC, CD45-PE, B220-PE, CD4-APC, CD8-APC, and CD105-APC antibodies were obtained from eBioscience. Lineage cocktail-APC, CD11c-APC.Cy7, α-BrdU-FITC and CD34-PE antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Arginase-FITC antibody was purchased from R&D systems (Minneapolis, MN, USA). For the flow cytometric analysis of Lineage-positive cells, the cells were stained with biotinylated Lineage cocktail obtained from BD Pharmingen, then labeled with streptavidin-V450 (BD Pharmingen). Data were collected using a FACSCalibur or LSRII-Green (both BD Pharmingen) and were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).