Isolation and Sorting of CNS Immune Cells

Mononuclear cells were isolated from the CNS as previously described and astrocytes, monocytes, and microglia were sorted as described before ^{10 (link),16 (link),17 (link)}. Isolated CNS cells were stained with fluorochrome-conjugated antibody to CD11b (M1/70, 1:100), CD45 (90, 1:100), Ly6C1 (HK1.4, 1:100), CD105 (N418, 1:100), CD140a (APA5, 1:100), CD11c (N418, 1:100), F4/80 (BM8, 1:50), O4 (O4, Miltenyi Biotec, 1:10), and CD19 (eBio1D3, 1:100). All antibodies were from eBioscience or BD Pharmingen, unless otherwise mentioned. Microglia were sorted as CD11b⁺ cells with low CD45 expression and low LY6C1 (CD11b⁺CD45^lowLy6C1^low), inflammatory monocytes were considered as CD45^hiCD11b⁺Ly6C1^hi. Astrocytes were sorted as CD11b^lowCD45^low Ly6C1^lowCD105^lowCD140a^lowCD11b^lowF4/80^lowO4^lowCD19^low after the exclusion of lymphocytes, microglia, oligodendrocytes, and monocytes.