CXCR3-deficient microglia display diminished levels for CD45 and CD11b during cuprizone diet. A. Ex vivo-prepared microglia of both genotypes were phenotyped using flow cytometry. B. Brain homogenates from wild type (WT) and CXCR3-/- mice after 3, 5, and 5.5 weeks of the cuprizone diet (n = 4 to 5 animals per group) were prepared and microglia subsequently analyzed for CD45 and CD11b expression. Using CD11b/CD45 gating, the majority of microglia were found in a distinct isolated CD11b+/CD45dim population in WT and CXCR3-/- animals (left encircled population). A small population of CD11b+/CD45high cells was slightly increased in WT compared to CXCR3-/- mice after 3 weeks (right encircled population, percentage of CD11b+/CD45high cells is given below, mean ± SEM). C. Flow cytometry histogram overlays show representative CD45/CD11b expression from freshly isolated microglia (CD11b+/CD45dim+high in WT (black line) and CXCR3-/- (red line) cuprizone-fed mice after 3 weeks. The dashed histogram represents the isotype control. Histograms were gated on microglial population according to forward/side scatter profile. CXCR3-/- animals displayed reduced surface expression levels for CD45 and CD11b compared with WT levels. D. Statistical analysis of the CD45 and CD11b level within the CD11b+/CD45dim microglia population of WT (black dots) and CXCR3-/- (red dots) mice over the course of the cuprizone experiment (mean fluorescence intensity, MFI). Fundamentally reduced MFI of CD45 (3 and 5 weeks) and CD11b (3 weeks) were documented in CXCR3-/- compared to WT microglia (**P <0.005, ***P <0.001).