Cd11c n418

Cells were released from culture plates using TrypLE and were stained after Fc-block (Invitrogen) with a panel of directly fluorochrome-conjugated mAbs against the following murine molecules (clone; source): Axl (R&D; Minneapolis, MN); CD4 (L3T4; eBioscience; San Diego, CA); CD8 (CD8b.2; Biolegend; San Diego, CA); CD11b (M1/70; eBioscience); CD11c (N418; eBioscience); CD19 (MCA1439F; AB Serotec); CD44 (IM7; eBioscience); CD45 (30-F11; BD); CD80 (16-10A1; BioLegend); CD206 (C068C2; BioLegend); Ly6C (AL-21; BD); Ly6G (1A8; BD, Franklin Lakes, NJ); Mertk (R&D). Experiments were performed on an LSR II flow cytometer and data were analyzed as previously described (25 (link)).

Cells from the colon and small intestine lamina propria were isolated as previously described^{8 (link)}. The cells were stimulated and stained as previously described^{8 (link)}. The following antibodies were used for surface staining of: CD3 (145-2C11, eBioscience); CD4 (L3T4, BD Difco); CD11b (M1/70, eBioscience); CD11c (N418, eBioscience); F4/80 (BM8, eBioscience); CD103 (M290, BD Difco); major histocompatibility complex (MHC) II (M5/114.15.2, BD Dico); TCR-γδ (eBioGL3, eBioscience); and NKp46 (29A1.4, eBioscience). Intracellular cytokine staining was performed using IL-17A (TC11-18H10, BD Difco) and IL-22 (IL-22JOP, eBioscience) antibodies. All antibodies were used at final concentration of 1 μg/ml. The cells were analyzed using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Leukocytes were gated using forward scatter (FSC) and side scatter (SSC), and within the leukocyte gates, the innate immune cells were identified as macrophages (MHCII⁺F4/80⁺CD103⁻CD11b⁺CD11c⁻) or dendritic cells (MHCII⁺F4/80⁻ CD103^+/−CD11b⁻CD11c⁺). For the lymphoid compartment, the leukocytes were gated using FSC and SSC. Within the lymphocyte gate, the populations were identified as T_H17 cells (CD3⁺CD4⁺IL-17⁺IL-22⁺), T_H22 cells (CD3⁺CD4⁺ IL-17⁻IL-22⁺), NKp46⁺ ILCs (including ILC3 and NK cells; CD3⁻CD4⁻NKp46⁺), LTi cells (CD3⁻CD4⁺NKp46⁻), γδ T cells (CD3⁺CD4⁻TCRγδ⁺) or CD3⁻CD4⁻NKp46⁻ cells.

Spleens and popliteal LNs were harvested at indicated times and prepared as previously described^{57 (link)}. Briefly, tissues were fixed in periodate-lysine-paraformaldehyde buffer and placed in 30% sucrose in PBS. Tissues were then embedded in OCT medium (Electron Microscopy Sciences), frozen in dry-ice cooled isopentane and sections were cut on a cryostat (Leica Microsystems). Sections were blocked in 5% sera and stained with the following antibodies to: CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), B220 (RA3–6B2, eBioscience), CD64 (polyclonal, R&D Systems), MHC II (I-A/I-E, M5/114.15.2, eBioscience), followed by the relevant secondary antibodies conjugated to fluorophores. Images were acquired using Leica SP8 microscope and analyzed using Imaris software (Bitplane).

Antibodies used for flow cytometry included B220 (RA3-6B2, Biolegend), CD3 (clone 145-2C11, BD Biosciences), CD4 (RM4-5, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), CD38 (90, eBioscience), CD44 (IM7, eBioscience), F4/80 (BM8, eBioscience), GL7 (GL7, BD Biosciences), Gr-1 (RB6, eBioscience), IgM (eB121-15F9, eBioscience), IL-17A (TC11-18H10, BD Biosciences), IL-17F (18F10, eBioscience), and IL-17RA (PAJ-17R, eBioscience).

In vivo labeling of T cells with fluorescent antibodies was performed as previously described (Turner et al., 2014). Briefly, mice were administered, PE- or Alexa 647-conjugated anti-CD90.2 (anti-Thy1.2; clone 53-2.1, BioLegend), or PE-conjugated anti-CD45.2 antibody (clone 104, Biolegend), intravenously, and 7–10 minutes later lungs were perfused, dissected and digested in RPMI1640 medium with collagenase D, DNAse and Trypsin inhibitor for 1 hour at 37°C. Mediastinal lymph nodes and spleen were dissected and manually disrupted to generate single cell suspensions. Cell suspensions were stained with fluorescent-conjugated antibodies for CD4 (clones RM4-5, BD Biosciences, and GK1.5, eBioscience), CD8 (clone 53-6.7, BioLegend), CD11a (clone M17/4, BioLegend),CD11b (M1/70 eBioscience), CD11c (N418, eBioscience), CD25 (PC61.5, eBioscience), CD45 (30-F11, BioLegend), CD69 (clone H1.2F3, eBioscience), CD86 (GL-1, Biolegend), CD103 (clone 2E7, eBioscience) and Anti-Mouse MHC Class II (I-A/I-E) (clone M5/114.15.2, eBioscience) was performed according to manufacturers’ protocol. Stained cells were analyzed using the BD LSRII flow cytometer and flow-jo software (Treestar, Ashland, OR). Absolute cell numbers were determined by flow cytometry using CountBright Absolute Counting Beads (Invitrogen) according to manufacturer’s protocol.