Multiparametric Flow Cytometric Analysis of Immune Cell Populations

Mononuclear cell suspensions were prepared as previously described ^{10 (link),16 (link),17 (link)}. Antibodies for flow cytometry were purchased from eBioscience or BD Pharmingen and used at a concentration of 1:100 unless recommended otherwise by the manufacturer. Mouse AHR antibody (IC6697G) and mouse FLT-1 antibody (FAB4711A) were from R&D Systems, VEGF-B (RM0008–6E72) and TGF-α (MF9) from Novus Biologicals, EGF Receptor (D38B1) and p-p65 (93H1) from Cell Signaling. Cells were then analyzed on a LSRII or MACSQuant flow cytometer (BD Biosciences and Miltenyi Biotec, respectively). As outlined in the individual figures, Th1 cells were defined as CD3⁺CD4⁺IFN-γ⁺IL-17⁻IL-10⁻Foxp3⁻, Th17 cells as CD3⁺CD4⁺IFN-γ⁻IL-17⁺IL-10⁻Foxp3⁻, Treg cells as CD3⁺CD4⁺IFN-γ⁻IL-17⁻Foxp3⁺, microglia as CD11b⁺CD45^lowLy6C^low, and pro-inflammatory monocytes as CD45^hiCD11b⁺Ly6C^hi.