Figure 3

Gating strategy to identify CD45+ hematopoietic immune cells that have accumulated in the central nervous system (CNS) of neonates and adult mice prior to onset of experimental autoimmune encephalomyelitis (EAE). (A) At day 10 post immunization with complete Freund adjuvant (CFA)/ myelin oligodendrocyte glycoprotein (MOG)_35-55, neonatal and adult mice were perfused and CNS cells isolated and prepared for flow cytometry. Cells were examined by side scatter (SSC) versus CD45 staining and three populations of CD45⁺ cells were identified; R1, CD45⁺ high SSC cells, which were Gr-1⁺ and represent granulocytes; R2, CD45⁺ hematopoetic immune cells, which were further divided into CD11b⁺ myeloid cells and CD11b^- non-myeloid cells containing lymphocyte subsets; and R3, CD45^dim microglia, which dimly express CD11b. Results are representative of three independent experiments. (B) Antigen-presenting cells (APC) within the CD45⁺CD11b⁺ cell population were identified by expression of Gr-1 to identify Gr-1⁺ monocytes, Gr-1^- monocytes, and CD11c⁺ DC. Histograms on the right represent staining for major histocompatibility complex (MHC) II using anti-I-A^b. (C) T cells were identified in the CD45⁺CD11b^- fractions by expression of CD3, CD4 and CD8. CD19⁺ B cells and DX5⁺ NK cells were identified in the CD45⁺CD11b^-CD3^- fraction. Plasmacytoid dendritic cells (PDC) were identified in the CD45⁺CD11b^-CD3^- by expression of B220 or PDCA1.