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Comprehensive analysis of mouse retinal mononuclear phagocytes

Nature Protocols volume 12, pages 1136–1150 (2017)Cite this article

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Abstract

The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRT–PCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies.

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Figure 1: Diagram illustrating the comprehensive microglia characterization procedure.
Figure 2: Retinal in vivo imaging of mice.
Figure 3: Immunohistochemical analyses of microglia in retinal cross-sections.
Figure 4: Immunohistochemical analyses of microglia in retinal flat mounts.
Figure 5: Flow cytometry analysis of retinal microglia from control mice and animals after light damage.
Figure 6: Real-time RT-PCR analyses of representative microglia activation marker genes.

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Acknowledgements

This work was supported by grants from the German Research Foundation (DFG; LA1203/6-2, LA1203/9-1, LA1203/10-1, and FOR2240), the ProRetina Foundation, the Hans and Marlies Stock Foundation, the Velux Foundation, Fight for Sight (1425/1426), Germany's Federal Ministry of Education and Research (BMBF; 03VP00272), the Graduate Program in Pharmacology and Experimental Therapeutics at the University of Cologne (in collaboration with Bayer), INSERM, Agence Nationale de la Recherche (ANR) MACLEAR (ANR-15-CE14-0015-01), Labex LifeSenses (ANR-10-LABX-65), the ANR (Investissements d'Avenir programme (ANR-11-IDEX-0004-02)), Carnot, the ERC (starting grant ERC-2007 St.G. 210345), and the Association de Prévoyance Santé de ALLIANZ.

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Author notes

  1. Anika Lückoff and Rebecca Scholz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Ophthalmology, Laboratory for Experimental Immunology of the Eye, University of Cologne, Cologne, Germany

    Anika Lückoff, Rebecca Scholz & Thomas Langmann

  2. Institut de la Vision, Sorbonne Universités, Université Pierre et Marie Curie, INSERM, Centre National de la Recherche Scientifique, Paris, France

    Florian Sennlaub

  3. Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, UK

    Heping Xu

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Contributions

A.L. and R.S. performed experiments, analyzed the data, and wrote the manuscript. F.S. and H.X. designed the research and corrected the manuscript. T.L. designed the research, obtained the funding, and finalized the manuscript. All authors read and approved the final manuscript. All authors have worked on and optimized this protocol.

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Correspondence to Thomas Langmann.

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Integrated supplementary information

Supplementary Figure 1 Gating strategy and staining controls for flow cytometry analysis.

Representative FACS plots of a healthy mouse retina (a). Doublets and dead cells were excluded from analysis by determination of a single and a live gate. Afterwards the number of CD11b+ cells can be analyzed (a). To determine a live gate, it is necessary to include an unstained control and heat-killed, ViobilityTM fixable dye stained control (b). To exclude nonspecific labeling of the CD11b+ antibody and to determine the CD11b+ gate, it is necessary to include Fluorescence minus one (FMO) controls without CD11b+ (FMO sample is labeled with FcR blocking reagent and ViobilityTM fixable dye) and isotype controls (c). (n=1 representative image). Male Balb/c mice with an age between 16 and 20 weeks were used for flow cytometry analysis. The use of mice in this experiment was approved by the governmental body responsible for animal welfare in the state of North Rhine-Westphalia, Germany with the permission numbers Az 84-02.05.20.12.158, Az 84-02-04-2014-A466 and Az 84-02-04-2015-A039 and followed the ARRIVE guidelines55.

Supplementary information

Supplementary Figure 1

Supplementary Figure 1. Gating strategy and staining controls for flow cytometry analysis. (PDF 343 kb)

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Lückoff, A., Scholz, R., Sennlaub, F. et al. Comprehensive analysis of mouse retinal mononuclear phagocytes. Nat Protoc 12, 1136–1150 (2017). https://doi.org/10.1038/nprot.2017.032

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