Fig 1. Migration efficiency of IV transferred monocytes and cMDM to acutely inflamed brain tissue is linked to monocyte-to-macrophage differentiation.

(A) MDM cultured from day 0 to day 28 were introduced IV (5×10⁶ cells) into recipient mice bearing LPS-induced acute neuroinflammaiton, and cMDM in vivo brain homing efficiency deceased as the in vitro cultivation time went up. Female gDNA = female mouse brain tissue genomic DNA (negative control). No cell IV = LPS injected control animal that received no cell IV transplant. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value <0.05. (B–D) Flow cytometry analysis on cMDM cell populations at different time points post in vitro cultivation. (B) Example of phenotype identification of cMDM using D5 cMDM. The green cluster reflects macrophage phenotype (CD11b⁺ F4/80^hi Ly6C^{low to neg}), blue cluster reflects Ly6c^low monocytes (CD11b⁺ F4/80^{low to med} Ly6C^{low to neg}), and yellow cluster reflects Ly6C^hi monocytes (CD11b⁺ F4/80^{low to med} Ly6C^high). (C) The ratio of macrophages (CD11b⁺ F4/80^high Ly6C^neg) and total monocytes (CD11b⁺ F4/80^{low to med} Ly6C^{low to high}), and (C) the ratio of Ly6C^hi monocytes (CD11b⁺ F4/80^med Ly6C^high CCR2⁺) and Ly6C^lo monocytes (CD11b⁺ F4/80^low Ly6C^low CCR2^{low to neg}) in culture were evaluated. (E) Phenotyping of freshly isolated EnMO by flow cytometry analysis. Expression level of CD115, CD11b, F4/80, and Ly6c were measured to determine the purity of the enriched cells. (F) Freshly isolated EnMO showed superior brain homing efficiency over cMDM. 5x10⁶ of EnMO (D0 EnMO), or MDM cultured for 2-, 5-, or 12- days (D2 cMDM, D5 cMDM, D12 cMDM, respectively) were infused IV to animals with acute neuroinlfammation, and the number of donor cells present in the LPS-injected brain hemisphere was quantified at 48 hour following cell IVI. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value all < 0.05.