Anti cd11c hl3

All antibodies were used FITC, PE, Cy3, PE-Cy5, PE-Cy7, APC, eFluor660 or biotin conjugated. The biotinylated mAb were revealed with Streptavidin-PE-Cy5. mAb were used at 0.1–1 µg/1×10⁶ cells in 100 µl PBS/2% FCS/0.02% NaN₃ (FACS buffer) or HBSS/0.3% BSA incubated on ice for 20–30 min. Antibodies used were: anti-CD16/CD32 (Fcγ III/II Receptor) (2.4G2), anti-CD90.1 (Thy1.1) (HIS51), anti-CD8α(Ly-2) (53–6.7), anti-CD3ε (145-2C11), anti-Vα2 T cell receptor (B20.1), anti-α4β7 (DATK32), anti-IA^b (AF6-120.1) and anti-CD11c (HL3) all from BD Biosciences (Heidelberg, Germany), anti-CCR9 (B cell hybridoma supernatant, kindly provided by Prof. R. Förster), anti-rat IgG (secondary for CCR9) (from Jackson ImmunoResearch Europe Ltd.), anti-CCR10 (248918), E-selectin/human IgG-Fc-Chimera (both from R&D) and polyclonal rabbit anti-human IgG (secondary for E-lig staining) (DakoCytomation, Hamburg, Germany).
All stainings were performed following a standard protocol except of E-lig staining. This staining was performed as described [16] (link). Control staining for E-lig and CCR9 was done with secondary Ab only, while corresponding isotype control mAb or fluorescence minus one (FMOs) were used as a control for all other stainings.
Data were acquired and analysed on a FACScan using CellQuestPro software (BD Biosciences) or on a FACS Canto II (Becton Dickinson) and analysed using FlowJo.

Commercially available ELISA Kits were used for the quantification of the cytokines IL-12p70, IL-23, IL-6 and IL-10 from cell culture supernatants. Immune cells were isolated from draining lymph nodes of immunized mice on day 3 or 7 as indicated and analzsed for cytokine production by qRT-PCR or flow cytometry. Intracellular cytokine staining was performed after stimulating cells with PMA and ionomycin (Sigma-Aldrich) in the presence of GolgiStop^® (BD Biosciences) for 4 h20 (link). Cells were fixed with 2% formaldehyde, permeabilized with saponin containing buffer and stained with fluorochrome-labeled antibodies directed against CD4 (Gk1.5, Biolegend) and IFN-γ (XMG1.2, eBioscience), IL-4 (11B11, eBioscience), IL-17A (TC11-18H10, BD Biosciences), IL-2 (JES6-5H4, eBioscience), IL-10 (JES5-16E3, eBioscience) or TNF (MP6-XT22, eBioscience). Fluorochrome-labeled anti-CD11c (HL3, BD Biosciences) antibodies were used for staining DC. Cells were analysed by flow cytometry (LSRII fow cytometer, BD Biosciences) and collected data were analysed by FLOWJO/FCS Express. Concentrations of indicated cytokines in BAL fluid were measured by Cytometric Bead Array (BD Biosciences, San Jose, CA, USA).

Flow cytometry was performed on mouse blood as previously described (63 (link)). Briefly, to obtain single-cell suspensions, spleen samples were ground between sterile frosted glass slides in 7 ml of red blood cell lysis buffer (0.15 mM NH₄Cl, 10 mM KHCO3, 0.1 mM disodium ethylenediaminetetraacetic acid, pH 7.2) and then filtered through sterile nylon mesh. Whole blood, obtained by cardiac puncture, was treated with red blood cell lysis buffer twice. Cell pellets were washed and resuspended in phosphate-buffered saline containing 2 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin. Fluorophore-conjugated antibodies with specificity to mouse cell antigens were as follows: anti-CD11b (M1/70) (BD Biosciences Cat# 550993, RRID:AB_394002), anti-CD11c (HL3) (BD Biosciences Cat# 561022, RRID:AB_2033997), anti-SiglecF (E50-2440) (BD Biosciences Cat# 552125, RRID:AB_394340), anti-Ly6G (1A8) (BD Biosciences Cat# 561236, RRID:AB_10611860), and anti-CD36 (JC63.1) (Cayman Chemical Cat# 10009870, RRID:AB_10342682). Live/dead fixable dead cell stains (ThermoFisher) were used to exclude dead cells. CD11b+/c+ cells were analyzed for CD36 expression. Paraformaldehyde-fixed cells were acquired using a Becton Dickinson (BD) LSR Fortessa flow cytometer (BD Biosciences, University of Alberta Flow Cytometry core) and analyzed with FlowJo (version 10) software (RRID:SCR_008520).

Tissues were excised from mice, minced and digested in Collagenase for 30 min. The samples were filtered through a 40-μm filter. Erythrocytes were removed with Lysing buffer (BD Biosciences). The samples were then incubated for 10 min with anti-CD16/CD32 blocking antibodies (BD Biosciences). The cells were stained with the following antibodies: anti-CD206 (C068C2, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD11b (M1/70, BioLegend), anti-CD45 (30-F11, eBioscience), anti-F4/80 (CI: A3-1, BD Bioscience), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD4 (GK1.5, BioLegend), anti-TCRβ (H57-597, BioLegend), anti-PD-1 (29F.1A12, BioLegend), and anti-CD44 (IM7, BioLegend). The samples were washed, incubated with 7-amino-actinomycin D (BD Biosciences) and then analysed on a FACSAria II (BD Biosciences).