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. 2021 Jun 16;218(8):e20210040. doi: 10.1084/jem.20210040

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© 2021 Milich et al.

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Figure 5. — In vivo validation of chemotaxis-inducing and inflammatory macrophages after SCI. (A) Immunohistochemical validation of chemotaxis-inducing and inflammatory macrophages and their temporal changes after SCI. Injured spinal cord tissue shows a steady increase in macrophages (CD11b⁺/CD63⁺) over time. APOE expression differentiates between chemotaxis-inducing (CD11b⁺/CD63⁺/APOE^lo) and inflammatory (CD11b⁺/CD63⁺/APOE^hi) macrophages. Chemotaxis-inducing macrophages are identified by white arrowheads, while inflammatory macrophages are identified by red arrowheads. Boxed regions on the left are shown in higher magnification on the right. Representative images from a total of three biological replicates per each time point. Scale bars in merged images are 300 µm, and scale bars in magnified images are 25 µm. Blue is DAPI to label nuclei. (B) Scatter plot of flow cytometry analysis and gating strategy (see Materials and methods for description) to quantify relative proportions of the two macrophage subtypes. After excluding neutrophils and lymphocytes, monocytes/macrophages were separated from microglia based on CD11b and CD45. After gating for CD63^hi macrophages, separation based on CD11b and APOE resulted in CD11b^med/APOE^lo (chemotaxis-inducing macrophages) and CD11b^hi/APOE^hi (inflammatory macrophages) clusters. (C) Quantification showed increased proportion of CD11b^hi/APOE^hi macrophages at 7 dpi compared with 1 dpi. n = 5 biological replicates at each time point for flow cytometry experiment. Macro, macrophage; Mono, monocyte.