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Updated: Sep 15, 2025
Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Published on: June 24, 2025
Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Yu-Qing Chen1, Jian-Xiong Gao2, Hua-Zheng Yan2
1Clinical Laboratory, the First Affiliated Hospital of Bengbu Medical University; Department of Biochemistry and Molecular Biology, School of Laboratory Medicine, Bengbu Medical University.
Abstract:
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- /lowCD68+CCR7+, CD11b+CD45- /lowCD68+CCR7-, CD11b+CD45highCD68+CCR7+, and CD11b+CD45highCD68+CCR7- cells can be identified as M1-like MG, M2-like MG, M1-like Mø, and M2-like Mø, respectively.
Insights
After spinal cord injury (SCI), distinguishing between microglia (MG) and macrophages (Mø) is challenging. This study presents a flow cytometry method to identify M1/M2-like MG and Mø phenotypes based on specific marker expression.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia (MG) and peripheral macrophages (Mø) are key immune cells in the spinal cord.
- Following spinal cord injury (SCI), MG activation and Mø infiltration are critical pathological processes.
- Differentiating pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes of MG and Mø is difficult due to overlapping markers.
Purpose of the Study:
- To develop a reliable flow cytometry (FCM) protocol for distinguishing M1/M2-like microglia and macrophages after SCI.
- To establish a method for analyzing the distinct roles of these immune cell phenotypes in SCI pathogenesis.
Main Methods:
- Utilized flow cytometry (FCM) to analyze cell surface and intracellular markers.
- Optimized FCM for stable detection of microglia and macrophages in the injured spinal cord.
- Identified specific cell populations using CD45, CD11b, CD68, and CCR7 marker expression profiles.
Main Results:
- Developed a protocol to differentiate M1/M2-like MG and Mø phenotypes.
- CD11b+CD45-/lowCD68+CCR7+ cells identified as M1-like MG.
- CD11b+CD45-/lowCD68+CCR7- cells identified as M2-like MG.
- CD11b+CD45highCD68+CCR7+ cells identified as M1-like Mø.
- CD11b+CD45highCD68+CCR7- cells identified as M2-like Mø.
Conclusions:
- The established FCM method enables precise identification of M1/M2-like microglia and macrophages post-SCI.
- This protocol aids in understanding the specific contributions of different immune cell phenotypes to spinal cord injury.
- Facilitates further research into targeted immunomodulatory therapies for SCI.
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