Fig 2. Recruited monocytes are mainly Ly6C^low populations.

(A) IVM images showing the expression of CX3CR1 and CCR2 on recruited monocytes in brain postcapillary venules of CX3CR1^gfp/+CCR2^rfp/+ mice 24 h after infection with 20x10⁶ C. neoformans (Green: CX3CR1, Red: CCR2). (B) Expression of Ly6C on recruited monocytes (arrow) in brain postcapillary venules of CX3CR1^gfp/+CCR2^rfp/+ mice 24 h after infection with 20x10⁶ C. neoformans. For labeling of Ly6C, mice were i.v. injected with 2 μg AF647-anti-Ly6C mAb through the tail vein 5 min before imaging. (C) Representative flow cytometry plots of CX3CR1⁺Ly6C^hi and CX3CR1⁺Ly6C^low monocyte subsets in the brain of naïve and infected C57BL/6 mice. Gated on CD45⁺ cells. The expression of CD11b on CX3CR1^- (filled) and CX3CR1⁺ (solid) populations was shown on the right. C57BL/6 mice were i.v. infected with 20x10⁶ C. neoformans and 24 h later brain leukocytes were isolated and analyzed by flow cytometry. (D) Flow cytometry analysis of the numbers of different subsets of cells recruited to the brain of C57BL/6 mice (n = 4 per group) 24 h after infection. Ly6C^hi monocytes were defined as CD45⁺Ly6G^-NK1.1^-CD11b⁺CX3CR1⁺Ly6C^hi, Ly6C^low monocytes as CD45⁺Ly6G^-NK1.1^-CD11b⁺CX3CR1⁺Ly6C^low, NK cells as CD45⁺NK1.1⁺, neutrophils as CD45⁺CD11b⁺Ly6G⁺. (E) A representative IVM image showing the recruitment of RFP⁺ monocytes in brain postcapillary venules of CCR2^rfp/rfp mice (CCR2 deficient) 24 h after i.v. infection with 20x10⁶ C. neoformans. See also S4 Video. (F) Flow cytometry analysis of the numbers of Ly6C^hi and Ly6C^low monocytes and neutrophils recruited to the brain of WT and CCR2^-/- mice (n = 4 per group) 24 and 48 h post i.v. infection with 20x10⁶ C. neoformans. Scale bars: 20 μm. Data are expressed as mean ± SEM and representative of 3 independent experiments. * p<0.05.