Multiparametric Flow Cytometry Analysis of Lung Immune Cells

Lung cell suspensions were adjusted to 1x10⁶ cells and suspended in PBS-azide (0.1%) containing fetal bovine serum (SFB, 5%). Fc receptors were blocked with anti-CD16/32 monoclonal antibody and then labeled with fluorophore-conjugated antibodies as previously described (37 (link)) Labeled antibodies (BD Biosciences) were used in the appropriate combination for the cell population to be analyzed. For lymphocytes, the following antibodies were used: anti-CD3, CD4, CD25, and Foxp3; for myeloid cells: anti-CD45, CD11b, CD11c, CD40, CD80, CD86, MHC-II, and F4/80. For ILCs characterization, lung leukocytes were first treated with an anti-mouse lineage cocktail (Biolegend) containing antibodies to CD3, Ly6G/Ly6C, CD11b, CD45R/B220, TER 119/erythroid cells, that react with T cells, B cells, monocytes, macrophages, NK cells, and erythrocytes. Intracellular staining was conducted using the eBioscience Transcription Factor staining kit and specific antibodies for IL-17, IL-4, IFN-γ, IL-22, IL-1β, IL-12, TNF-α, IL-6, TGF-β, IL-10, FoxP3, IDO-1, and AhR. Supplementary Table 1 lists the monoclonal antibodies used in flow cytometry assays. Cells were run on FACSCantoII (BD Biosciences) and a minimum of 50,000 events was acquired using FACSDiva software (BD Biosciences). Cells were analyzed using FlowJo software (Tree Star).