Fig. 2

Experimental Design, Gating Strategy, and Flow Analysis of Innate and Adaptive Immune Cells from the Midbrain. (A) Male Line 61 mice (12–15 weeks old) were bilaterally injected in the striatum with monomer (10 µg) or PFF (10 µg). Two weeks after PFF injection, VH (DMSO) or AZD1480 (25 mg/kg/day) was administered by oral gavage for an additional four weeks for a total of six weeks. (B) Mononuclear cells were isolated from midbrains. The gating strategy of flow cytometry analysis was as follows: CD45^MedCD11b⁺ microglia, CD45^HiCD11b⁺ macrophages, CD45⁺CD11b⁺CD11c⁺ dendritic cells (DCs), CD45⁺CD11b⁻CD4⁺ T-cells, CD45⁺CD11b⁻CD8⁺ T-cells, and CD45⁺CD11b⁻CD19⁺ B-cells. Representative gating plots of MHC Class II expression in microglia, macrophages, and DCs are shown. Fluorescence minus one (FMO) was used as a control. (C) Six weeks post injection, mononuclear cells were isolated from the midbrains of monomer (n = 4), PFF + VH (n = 8), or PFF + AZD1480 (25 mg/kg/day) (n = 12) mice, then subjected to flow cytometry analysis. Absolute numbers of total CD45⁺ immune cells, CD45^MedCD11b⁺ microglia, CD45^HiCD11b⁺ macrophages, CD45⁺CD11b⁺CD11c⁺ DCs, and CD45⁺CD11b⁻ lymphocytes are shown as mean ± SD. (D) Absolute numbers of MHC Class II-positive CD45^MedCD11b⁺ microglia, macrophages, and DCs are shown as mean ± SD. (E) Absolute numbers of CD4⁺ T-cells, CD8⁺ T-cells, and CD19⁺ B-cells are shown as mean ± SD. Statistical significance was determined by ordinary one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001