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Updated: Jun 10, 2025
Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Published on: June 22, 2017
Visualizing the spatial organization of monocytes, interstitial macrophages, and tissue-specific macrophages in situ
Maxime Petit1, Eléonore Weber-Delacroix1, François Lanthiez1
1Sorbonne Université ́, Inserm U1135, CNRS ERL 8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France.
Abstract:
Tissue-resident mononuclear phagocytes (MPs) are an abundant cell population whose localization in situ reflects their identity. To enable assessment of their heterogeneity, we developed the red/green/blue (RGB)-Mac mouse based upon combinations of Cx3cr1 and Csf1r reporter transgenes, providing a complete visualization of their spatial organization in situ. 3D-multi-photon imaging for spatial mapping and spectral cytometry employing the three markers in combination distinguished tissue-associated monocytes, tissue-specific macrophages, and three subsets of connective-tissue-associated MPs, including CCR2+ monocyte-derived cell, CX3CR1+, and FOLR2+ interstitial subsets, associated with distinct sub-anatomic territories. These populations were selectively reduced by blockade of CSF1, CSF2, CCR2, and CX3CR1 and efficiently reconstitute their spatial distribution after transient myelo-ablation, suggesting an autonomous regulatory environment. Our findings emphasize the organization of the MP compartment at the sub-anatomic level under steady-state conditions, thereby providing a holistic understanding of their relative heterogeneity across different tissues.
Insights
Researchers developed the RGB-Mac mouse to visualize tissue-resident mononuclear phagocytes (MPs). This tool reveals MP heterogeneity and spatial organization within tissues, offering new insights into immune cell distribution.
Area of Science:
- Immunology
- Cell Biology
- Developmental Biology
Background:
- Tissue-resident mononuclear phagocytes (MPs) are crucial immune cells residing in various tissues.
- Understanding the heterogeneity and spatial organization of MPs is essential for their functional roles.
- Existing methods lack the resolution to fully map MP diversity in situ.
Purpose of the Study:
- To develop a novel reporter mouse model for comprehensive in situ visualization of MP heterogeneity.
- To spatially map distinct subsets of mononuclear phagocytes within different tissue microenvironments.
- To investigate the regulatory mechanisms governing MP localization and reconstitution.
Main Methods:
- Generation of the red/green/blue (RGB)-Mac mouse model using Cx3cr1 and Csf1r reporter transgenes.
- Application of 3D-multi-photon imaging for spatial mapping of MP populations.
- Utilizing spectral cytometry with three markers to distinguish and quantify MP subsets.
Main Results:
- The RGB-Mac mouse enabled complete visualization of MP spatial organization in situ.
- Distinct MP subsets, including tissue-associated monocytes, tissue-specific macrophages, and interstitial MPs (monocyte-derived, CX3CR1+, FOLR2+), were identified.
- These populations showed selective reduction upon blockade of CSF1, CSF2, CCR2, and CX3CR1, and demonstrated efficient reconstitution post-myelo-ablation.
Conclusions:
- The RGB-Mac mouse is a powerful tool for assessing MP heterogeneity and spatial distribution.
- MP organization occurs at a sub-anatomic level, highlighting distinct territories for different subsets.
- Findings suggest an autonomous regulatory environment governing MP compartment organization and dynamics.
