Distinguishing Microglia and Macrophages

Brain single-cell suspensions from 9-mo-old WT mice were stained with Ghost Dye violet 510 (1:1,000; Tonbo Biosciences) and anti–CD16/CD32 antibody (1:100; BD Biosciences) followed by anti-CD11b (1:100; BioLegend), anti-CD45 (1:100; BioLegend), anti-CD11c (1:50; BioLegend), anti-TMEM119 (1:200; Abcam), and anti-CCR2 (1:100; BioLegend). CD11b⁺ cells were gated from single/live cells followed by subsequent gating of CD11b⁺CD45^low as microglia and CD11b⁺CD45^high as macrophages. To distinguish microglia and macrophages, the CCR2 marker expressed by blood-derived macrophages but not by microglia was used (3 (link), 16 (link)). To further distinguish these two cell types, the microglia-specific marker Tmem119 (17 (link)) was also included. The CD11b⁺CD45^high cells that express CCR2 but not Tmem119 were confirmed as macrophages, while the CD11b⁺CD45^low cells that all express Tmem119 but do not express CCR2 were confirmed as microglia. FMO negative controls were included to confirm the specificity of CD11c staining in CD11b⁺CD45^low microglial populations. Brain CD45⁻ cells that mainly contain nonimmune cells (e.g., neurons, astrocytes, and oligodendrocytes) that do not express CD11c were also included as negative controls to further validate the specificity of this strategy.