Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry

In a healthy spinal cord, peripheral infiltrating macrophages are almost undetectable, and microglia (MG) participate in maintaining the stability of the spinal cord microenvironment by phagocytosis, clearing cellular debris, and producing neurotrophic factors. After spinal cord injury (SCI), MG are activated, and peripheral immune cells infiltrate into the injured spinal cord. Among these immune cells, activated MG and peripheral infiltrating macrophages (Mø) play crucial roles in the pathological process of SCI. These cells can be distinguished into pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes; however, distinguishing them is challenging due to their similarity in morphology and many cellular markers. Flow cytometry (FCM), a widely used technique in the biomedical field, can simultaneously detect multiple cellular parameters such as cell size, particle size, cell surface, and intracellular markers in a single experiment. Over years of research, FCM was attempted to identify MG and Mø in spinal cords and was continuously optimized to ultimately develop a stable detection method. This method allows for the identification of M1/M2-like MG and M1/M2-like Mø after SCI by detecting the expression profiles of CD45, CD11b, CD68, CCR7, and other markers. In this protocol, CD11b⁺CD45^{- /low}CD68⁺CCR7⁺, CD11b⁺CD45^{- /low}CD68⁺CCR7^-, CD11b⁺CD45^highCD68⁺CCR7⁺, and CD11b⁺CD45^highCD68⁺CCR7^- cells can be identified as M1-like MG, M2-like MG, M1-like Mø, and M2-like Mø, respectively.