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Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes
Published on: June 17, 2020
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Comprehensive analysis of mouse retinal mononuclear phagocytes
Anika Lückoff1, Rebecca Scholz1, Florian Sennlaub2
1Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Cologne, Germany.
Nature Protocols
|May 5, 2017
Summary
This study introduces a comprehensive 3-day workflow to analyze retinal mononuclear phagocytes in mouse models of retinal disease. The method enables detailed characterization of immune cell responses in various ocular conditions.
Area of Science:
- Immunology
- Ophthalmology
- Cell Biology
Background:
- The innate immune system, particularly retinal mononuclear phagocytes (microglia, macrophages, monocytes), plays a crucial role in degenerative and inflammatory retinal disorders like age-related macular degeneration (AMD).
- Previous studies on these cells in mouse models have been limited by single detection methods, hindering a complete understanding of their behavior.
Purpose of the Study:
- To present a comprehensive and standardized analysis strategy for characterizing retinal mononuclear phagocytes in vivo and in situ.
- To provide a rapid (3-day) protocol applicable to various mouse models of retinal disease.
Main Methods:
- Scanning laser ophthalmoscopy using MacGreen reporter mice.
- Quantitative analysis of Iba1-stained retinal sections and flat mounts.
- CD11b-based retinal flow cytometry and qRT-PCR for microglia markers.
Main Results:
- The workflow successfully characterized retinal mononuclear phagocytes in models of choroidal neovascularization (CNV), light-induced injury, and inherited retinal degeneration.
- Standardized steps allow for detailed in vivo and in situ analysis of immune cell behavior.
Conclusions:
- This comprehensive workflow offers a valuable tool for studying immune responses in retinal diseases.
- The protocol can aid in evaluating immunomodulatory therapies by documenting immune cell dynamics in various mouse models.
