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Fig. 1 | Journal of Neuroinflammation

Fig. 1

From: Phagocytosis converts infiltrated monocytes to microglia-like phenotype in experimental brain ischemia

Fig. 1

Stroke-induced monocyte trafficking to the brain. a Experimental timeline. Middle cerebral arterial occlusions (MCAO) were performed either 3 days (d) or 7d prior to killing. Spleens were removed (SPX) 3d prior to killing the mice. GFP+ splenocytes (1–2 × 107) were infused retro-orbitally into asplenic mice at 1d prior to killing (or 2d post-splenectomy) so that GFP+ splenocytes circulated either during post-stroke days 2 and 3 for 3-day survival experiments or during post-stroke days 6 and 7 for 7-day survival experiments. b Flow cytometry and counts for GFP+ cells in the brain that were isolated by Percoll gradient at either 3 days (n = 6–7) and 7 days (n = 6), as well as for GFP+ cells isolated with debris removal method at 7 days (n = 6–7). Live cells were gated as negative for propidium iodine (PI) staining. c Gating strategy to identify mononuclear phagocytes at 7 days. R1–R4 are subsets of CD11b+ cells based on GFP expression with R1 and R3 being GFP− cells, whereas R2 and R4 were infiltrating GFP+ cells. Both R1 and R2 were cells derived from the contralateral hemisphere, whereas R3 and R4 were derived from the ipsilateral hemisphere. To identify mononuclear phagocytes with the R1–R4 subsets, a refined analysis removed NK cells and neutrophils based on NK1.1 and Ly6G expression, respectively. The resulting CD11b+/CD45+/NK1.1−/Ly6G− population was subdivided into R1’–R4’ subsets using the schema as the R1–R4 subsets. d Counts for CD11b+ cells in R1–R4. e Counts of CD11b+/CD45+/NK1.1−/Ly6G− microglia and MDMs in R1’–R4’. Statistical significance was assessed with two-way ANOVA for isolation with Percoll gradient method (** indicating p < 0.01), Student t-test for isolation with debris removal (**** indicating p < 0.0001), and with one-way ANOVA with post hoc Bonferroni comparison test for cell counts in d and e (with *, **, and ****, respectively, indicating p < 0.05, 0.01, and 0.0001)

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