Macsquant flow cytometer

Assessment of MSC presence in the CNS was performed as previously described [31 (link), 32 (link)]. Briefly, mice were perfused with ice-cold PBS, and the brain and spinal cord were removed and incubated with collagenase type III and DNase in PBS. Tissues were then homogenized and loaded on a Percoll gradient to enrich CNS infiltrates. Flow cytometry analysis for detection of stained MSCs, as well as DCs, encephalitogenic and regulatory T cells, was performed. Similarly, the kidneys, lungs, spleen, gut, and heart were homogenized and analyzed for MSC presence. 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. Cells were analyzed on an 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⁻IL-10^−/+Foxp3⁺, and pro-inflammatory monocytes as CD45^hiCD11b⁺Ly6C^hi.

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.