Figure 3

Flow cytometry and qRT/PCR analyses of immune cell infiltrates. (A) Flow cytometry gating strategy for macrophages (CD11b⁺CD45^hiLy6G^-), microglia (CD11b⁺CD45^loLy6G^-), and neutrophils (CD11b⁺CD45^hiLy6G⁺). (B) Macrophages or microglia were further characterized as alternatively activated (M2) CD206⁺Ym1⁺Arg1⁺ or (C) classically activated (M1) CD86⁺MHCII⁺iNOS⁺. (D) Flow cytometry gating for T cell subsets: Th1 = CD4⁺IFN-γ⁺, Th17 = CD4⁺IL17⁺, Treg = CD4⁺CD25⁺FOXP3. (E, F, G) Flow cytometry of cells isolated from pooled brain and spinal cord showed that the only significant difference in total numbers of T cell subtypes isolated from the CNS at 21 dpi was a decrease in the ratio of IFN-γ⁺ to IL-17⁺ cells in the astroglial CXCL10 knockout mice (n = 3 mice/group, P = 0.0063). (H, I) No significant differences between astroglial CXCL10 knockout and control mice in total numbers of macrophages, microglia, and neutrophils (H) or M1 and M2 subtypes of macrophages and microglia (I) isolated from the CNS at 21 dpi (n = 3). (J) qRT/PCR of spinal cord tissue isolated at 14 dpi normalized to the housekeeping gene HSP90; CXCR3 expression was significantly upregulated in astroglial CXCL10 knockout mice compared to controls (P = 0.0157). IFN-γ, FOXP3, RORγt, iNOS, and arginase-1 mRNA levels were not significantly different between astroglial CXCL10 knockout and control mice at 14 dpi (n = 6 mice/group). Vertical bars = SEMs.