Cd11b percpvio700

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.

Nonspecific antibody binding was blocked with FcR Blocking Reagent (Miltenyi Biotec). Antibodies used for cell-surface staining were CD45-APC-Cy7, F4/80-Pacific Blue (BioLegend, Fell, Germany), Gr-1-APC, CD11b-PerCPVio700, Siglec-f-PE, MHC-Class II-FITC, CD11c-FITC and CD49b-PE (Miltenyi Biotec). Cells were blocked, stained with antibodies according to the manufacturer's protocol, and fixed for 20 min in 2.25% formaldehyde [75] (link). Cells were washed with autoMACS Buffer and analysed using the MACSQuant Analyzer (Miltenyi Biotec). Gating strategy for identification of different subsets was based on Hackstein et al. [77] (link). Briefly, cells were gated based on scatter light (FSC, SSC) characteristics and viable singlet leukocytes were identified by CD45 expression. Out of the CD45-positive cells, neutrophils were identified by Gr-1^bright CD11b^bright expression. Out of the neutrophil negative fraction, eosinophils were identified by Siglec-f⁺ MHC-Class-II^neg expression. Out of the neutrophil and eosinophil negative fraction macrophages were identified as Siglec-f⁺⁺ F4/80⁺ double positive cells. Total dendritic cells were identified as CD11c⁺ Siglec-f^neg CD49b^neg leukocytes. Cell numbers of subsets were calculated per organ.