Cd11c percp cy5

Cell surface markers were measured using a flow cytometer (BD Biosciences, San Jose, CA, USA). The following antibodies were used: Linage-FITC, HLA-DR-APC, CD123-PE, and CD11c-PerCP-Cy5.5 (Biolegend, San Diego, CA, USA). The Fixable Viability Dye eFluor 780 (eBiosciense, San Diego, CA, USA) was used to exclude dead cells from the analysis. Magnetic beads coated with anti-rat and -mouse antibodies were used as positive and/or negative controls for calculating compensation (BD Biosciences, San Jose, CA, USA). We characterized mDC as Linage⁻HLA-DR⁺CD11c⁺, while pDC were characterized as Linage⁻HLA-DR⁺CD123⁺. Data was exported and analyzed using FlowJo software v7.6.1.

Isolation and analysis of skin lymphocytes [18 (link), 27 (link)] were carried out as described. Briefly, after removing fur with an electric groomer, trunk skin was separated from subcutaneous fat tissue, cut to small pieces and, for lymphocyte analysis, digested for 45 min. at 37°C in RPMI 1640 containing collagenase XI (4000 U/ml), hyaluronidase (260 U/ml) and DNase (0.1mg/ml), all from Sigma-Aldrich. Cells were harvested by filtration, washed and stained, first for surface markers with mAbs CD45.2-PerCP, CD3-APC-eFluor780, CD4-FITC, CD25-PE, CTLA4-APC, ICOS-PE-Cy7, and live/dead-eFluor506, then fixed and permeabilized and stained with FoxP3-eFluor450, all from eBioscience. For DC analysis, digestion was with 150μg/ml Liberase and 120μg/ml in HBSS and cells were stained with mAbs CD11c-PerCPCy5.5 and CD103-PE (both BioLegend) and, after fixation and permeabilization, with anti-langerin-Alexa488 (clone 929F3.01, Dendritics).

Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45‐vioblue 450, CD8‐FITC, CD19‐APC (Tonbo, San Diego, CA), HLA‐DR‐FITC, CD3‐viogreen, CD103‐PE (Miltenyi Biotec, Auburn, CA, USA), CD11c‐PerCp‐Cy5.5, CD69‐PE‐Cy7 (Biolegend, San Diego, CA), CD103‐PE‐Cy7, CD4‐PE (eBiosciences, San Diego, CA, USA), CD56‐APC (BD Pharmingen, San Diego, CA, USA). Dead cells were excluded with 7AAD (Southern Biotech) or zombie dye yellow staining (Biolegend). Analysis was performed on 8‐color MACSQuant 10 (Miltenyi biotech) or Gallios (Beckman Coulter) flow cytometers and data were analyzed with FlowJo software (Tree Star, Inc. Ashland, OR, USA). Expression of surface markers is shown as percentage of positive cells and MFI. Fluorescence minus one (FMO) strategy was used to establish appropriate gates. Comparisons between FMO and isotype controls showed no differences. For tissue dendritic cell (DC) quantification, DCs were identified using flow cytometry as CD45⁺, CD3‐, CD19‐, CD56‐, HLA‐DR^high, CD11c⁺ cells as described before (Rodriguez‐Garcia et al., 2017), and the cell number was normalized to tissue weight.

Cells were incubated with Fc Block (1 μg/10⁶ cells; 2.4G2; BD Biosciences) for 15 min. Extracellular staining was performed for 30 min on ice using the following antibodies: CD4-BV421 (RM4-5; BioLegend, San Diego, CA, USA), CD45-BV510 (30-F11; BioLegend), CD3-APC (17.A2, BioLegend), CD25-PE-Cy7 (PC61; BioLegend), CD8 PerCPCy5.5 (53-6.7; BioLegend), B220-APC-Cy7 (RA3-6B2; BD Biosciences), CD11b-PE-Cy7 (M1/70; BioLegend), Ly6C-PE (HK1.4; BioLegend), Gr1-APC-Cy7 (RB6-8C5; BioLegend), IA/IE-BV421 (M5/114.15.2; BioLegend), F4/80-FITC (BM8; BioLegend), CD11c-PerCP-Cy5.5 (N418; BioLegend).
After staining for extracellular proteins, cells were fixed in 4% paraformaldehyde (PFA, pH 7.4) and permeabilized using 0.1% saponin buffer containing 0.1% bovine serum albumin. For intracellular cytokine detection, interferon-γ (IFNγ)-BV421 (XMG 1.2, BioLegend), interleukin (IL)-10-PE (54902, BD Bioscience), and IL-17A-AF647 (TC11-18H10, BioLegend) antibodies were used.
Flow cytometry was performed on a BD FACS Canto II (BD Biosciences) and analyzed using FlowJo software version 10.1 (Treestar Inc., Ashland, OR, USA).