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Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Published on: June 22, 2017
Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Elodie Martin1, Mohamed El-Behi1, Bertrand Fontaine2
1Inserm U 1227, CNRS UMR 7225; Sorbonne Universités, UPMC, University of Paris; Institut du Cerveau et de la Moelle épinière, ICM.
Abstract:
Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.
Insights
This study differentiates central nervous system (CNS) macrophages, microglia and monocyte-derived macrophages (MDM), using flow cytometry. This method aids understanding of macrophage roles in neurological diseases and drug efficacy.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Macrophages play critical roles in central nervous system (CNS) pathologies.
- Two main CNS macrophage populations exist: resident microglia and infiltrating monocyte-derived macrophages (MDM).
- Distinguishing these populations historically lacked consensus on markers.
Purpose of the Study:
- To present a protocol for distinguishing microglia from MDM in the CNS.
- To enable detailed characterization of CNS macrophage subpopulations.
- To facilitate research into macrophage functions in neurological disease models.
Main Methods:
- Density gradient centrifugation for cell isolation.
- Flow cytometry analysis utilizing CD11b and CD45 marker expression.
- Characterization of specific macrophage subpopulations (microglia: CD11b+CD45med; MDM: CD11b+CD45high).
Main Results:
- Successfully differentiated microglia from MDM based on CD45 expression levels.
- Demonstrated the utility of density gradient centrifugation and flow cytometry for CNS macrophage analysis.
- Established a reproducible method for characterizing these distinct cell populations.
Conclusions:
- The described protocol provides a reliable method to distinguish and analyze CNS macrophage subpopulations.
- This technique enhances the understanding of macrophage involvement in neurological diseases.
- The method is applicable for evaluating therapeutic interventions targeting CNS macrophages.
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