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Updated: Mar 26, 2026
Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes
Published on: June 17, 2020
Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by
E G O'Koren1, R Mathew1, D R Saban1,2
1Department of Ophthalmology, Duke University School of Medicine, Durham, NC, USA.
Abstract:
The recent paradigm shift that microglia are yolk sac-derived, not hematopoietic-derived, is reshaping our knowledge about the isolated role of microglia in CNS diseases, including degenerative conditions of the retina. However, unraveling microglial-specific functions has been hindered by phenotypic overlap of microglia with monocyte-derived macrophages. The latter are differentiated from recruited monocytes in neuroinflammation, including retina. Here we demonstrate the use of fate mapping wherein microglia and monocyte-derived cells are endogenously labeled with different fluorescent reporters. Combining this method with 12-color flow cytometry, we show that these two populations are definitively distinguishable by phenotype in retina. We prove that retinal microglia have a unique CD45(lo) CD11c(lo) F4/80(lo) I-A/I-E(-) signature, conserved in the steady state and during retinal injury. The latter was observed in the widely used light-induced retinal degeneration model and corroborated in other models, including whole-body irradiation/bone-marrow transplantation. The literature contains conflicting observations about whether microglia, including in the retina, increase expression of these markers in neuroinflammation. We show that monocyte-derived macrophages have elevated expression of these surface markers, not microglia. Our resolution of such phenotypic differences may serve as a robust way to help characterize isolated roles of these cells in retinal neuroinflammation and possibly elsewhere in CNS.
Insights
Microglia, crucial for CNS health, were previously hard to distinguish from other cells. This study identifies a unique cell surface marker signature for retinal microglia, aiding research into neurodegenerative diseases.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the resident immune cells of the central nervous system (CNS), are increasingly recognized for their role in neurodegenerative diseases.
- Distinguishing microglia from infiltrating monocyte-derived macrophages in the CNS, particularly the retina, has been challenging due to overlapping phenotypes.
- Recent understanding suggests microglia originate from yolk sac progenitors, distinct from hematopoietic stem cells.
Purpose of the Study:
- To develop a method for definitively distinguishing between microglia and monocyte-derived macrophages in the retina.
- To identify a unique phenotypic signature for retinal microglia.
- To clarify the expression patterns of specific surface markers on microglia versus monocyte-derived macrophages during retinal inflammation and injury.
Main Methods:
- Utilized endogenous fluorescent reporters for fate mapping of microglia and monocyte-derived cells.
- Employed 12-color flow cytometry to analyze cell populations.
- Investigated cell phenotypes in various models, including light-induced retinal degeneration and whole-body irradiation/bone-marrow transplantation.
Main Results:
- Established a definitive phenotypic distinction between retinal microglia and monocyte-derived macrophages.
- Identified a unique signature for retinal microglia: CD45(lo) CD11c(lo) F4/80(lo) I-A/I-E(-).
- Demonstrated that monocyte-derived macrophages, not microglia, exhibit elevated expression of these markers during retinal injury.
Conclusions:
- The identified phenotypic signature provides a robust method for isolating and characterizing retinal microglia.
- This distinction is crucial for understanding the specific roles of microglia in retinal neuroinflammation and degenerative diseases.
- The findings may be applicable to distinguishing these cell types in other CNS inflammatory conditions.
