Abstract
The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.
Original language | English (US) |
---|---|
Article number | e61348 |
Pages (from-to) | 1-20 |
Number of pages | 20 |
Journal | Journal of Visualized Experiments |
Volume | 2020 |
Issue number | 160 |
DOIs | |
State | Published - Jun 2020 |
Funding
JAL was supported by NIH grant K08EY030923; CMC was supported by a K01 grant (5K01AR060169) from the NIH National Institute of Arthritis and Musculoskeletal Diseases and a Novel Research Grant (637405) from the Lupus Research Alliance.
ASJC Scopus subject areas
- General Chemical Engineering
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology
- General Neuroscience