Cd11c af700

Intestinal and mesenteric lymph node immune cell populations were characterized by flow cytometry. For intracellular cytokine staining, cells were incubated in T cell medium (RPMI, 10% FBS, 1 mM sodium pyruvate, 2 mM GLUTamax, 1× nonessential amino acids, 0.1 mM 2-β-mercaptoethanol, 10 mM HEPES, 1% penicillin/streptomycin) + 1× Cell Stimulation Cocktail with Protein Inhibitors (eBioscience) for 3 h at 37 °C before staining. Nonviable cells were stained using Live/Dead Fixable Aqua. Cells were permeabilized with CytoFix/CytoPerm (BD), followed by intracellular staining in PermBuffer (eBioscience).
All antibodies were purchased from eBioscience unless otherwise indicated. Antibodies used were CD45-SB600, TCRb-APC-Cy7, MHCII-FITC, CD4-PE-Cy7, CD8a-AF700, CD8b-PE, IFNg-PerCP-Cy5.5, TNFa-APC, IL-17A-bv421 (BioLegend), CD3e-FITC, TCRg-FITC, B220-FITC, MHCII-APC-e780, CD11b-PerCP-Cy5.5, CD11c-AF700, CD64-APC (BioLegend), SiglecF-PE (BD), Ly6G-Pe-Cy7 (BD) and F4/80-e450. Sample data were acquired with an Attune NxT flow cytometer coupled with an Attune CytKick Max autosampler. Data were analyzed using FlowJo v.10. Antibody details and concentrations are included in Supplementary Table 1.

BAL was centrifuged to collect cell pellets and supernatant stored for protein measurements (Bradford assay). Cells were counted by Multisizer (Beckman Coulter Indianapolis IN). A total of 30,000 cells were spun for cytospin, stained with KWIK-DIFF (Thermo Scientific) and morphologically analyzed for cell type. A total of 200,000 cells (or remaining if insufficient) were immunostained for flow cytometry using the following antibodies: CD206-PE (Biolegend 141706), Ly6C-PerCP/Cy5.5 (Biolegend 128012), F4/80-PE/Cy5 (Biolegend 123114), CD11b-APC (Biolegend 101212), and CD11c-AF700 (Biolegend 117320). Manufacturer protocols were followed including 10-min Fc block (TruStain FcBlock Biolegend 101320) followed by 30-min antibody incubation at manufacturer suggested concentrations. Viability dye-eFluor780 (eBiosciences 65–0865–14 San Diego, CA) was incubated after antibodies for 30 min and washed out before paraformaldehyde fixing cells. Gallios flow cytometer (Beckman Coulter Indianapolis IN) was used to analyze cell fluorescent expression. Data was analyzed using Kaluza software, and all BAL cells were gated on F4/80 and viability to examine the macrophage population (Beckman Coulter Indianapolis IN). Gating strategy is shared in Figure 1.