(B) PE vs APC-Cy7 plot for clean bead gate

(B) PE vs APC-Cy7 plot for clean bead gate. cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced quantity of fluorophores on most microscopes. We describe the use of multi-parametric circulation cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter circulation cytometry can be utilized NS-018 for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies. Introduction The innate immune system includes multiple cell types that activate match activation and inflammation. Innate immune cells include natural killer (NK) cells, mast cells, basophils, eosinophils, neutrophils, and mononuclear phagocytes. Mononuclear phagocytes, which are composed of monocytes, macrophages, and dendritic cells, have been implicated in the pathophysiology of multiple ophthalmic conditions including uveitis, diabetic retinopathy, and age-related macular degeneration (AMD)1 . In this protocol, we will focus on the identification of mononuclear phagocytes using multi-parameter circulation cytometric analysis in a mouse model of neovascular AMD2 . This protocol is usually flexible for mouse models of diabetic retinopathy and/or uveitis, but more considerable ocular dissection is recommended because of the systemic nature of these diseases. Mononuclear phagocytes express overlapping cell surface markers. Long-lasting tissue resident macrophages and microglia originate from the yolk sac-derived erythromyeloid progenitor3 , while recycling macrophages and dendritic cells differentiate from your bone marrow-derived macrophage dendritic cell progenitor4. Mouse cell surface markers common to monocytes, macrophages, and dendritic cells include CD45, CD11b5, F4/806, Cx3cr17, and the intracellular marker Iba18. In order to overcome this challenge, transcriptomic analysis of macrophages, monocytes, and dendritic cells from multiple tissues defines CD64 as a macrophage-specific cell surface marker6. Macrophages have been explained in the iris, choroid, ciliary body, and optic nerve in healthy eyes3. Alternatively, dendritic cell identification is usually more difficult; the most specific method of dendritic cell identification requires fate mapping using the Zbtb46-GFP reporter mouse9 . Impartial of this reporter line, expression of CD11c and MHCII in conjunction with the absence of CD64 can identify potential dendritic cells6, 10. Dendritic cells have been recognized in cornea, conjunctiva, iris, and choroid in normal eyes11 . Microglia are specialized macrophages located in the retina, guarded by the blood-retinal barrier, and derived from yolk sac progenitor cells12 . As a result, retinal microglia can be differentiated from monocyte-derived macrophages by their dim levels of CD45 expression13 and high levels of Tmem119, which is usually available as a circulation cytometry antibody14 . Upon microglia activation, however, CD45 can be up-regulated15 and NS-018 Tmem119 may be down-regulated3 , demonstrating the complexity of microglia biology and this is likely relevant in both AMD and its mouse model. Finally, monocytes can be divided into at least two subtypes, including classical and non-classical. Classical monocytes display CCR2+ Ly6Chigh CX3CR1low expression, and non-classical monocytes demonstrate CCR2? Ly6Clow CX3CR1high markers5 . Due to the necessity of the quantitative analysis of marker expression, i.e., high versus low/dim levels, multi-parameter circulation cytometry is the ideal method for discrimination between monocytes, macrophages, microglia, and dendritic cells in the eye and other tissues. Additional advantages include the identification of sub-populations, the ability to use fluorescence-activated cell sorting (FACS) to sort cell populations for transcriptomic or proteomic analysis, and fate mapping. The major disadvantage of multi-parameter circulation cytometry is the lack of tissue architecture. This can be overcome by the ophthalmic dissection into the numerous ocular subcompartments: cornea, conjunctiva, iris, lens, retina, and choroid-sclera complex. Additionally, confirmatory immunofluorescence imaging can be performed, but is limited by the number of markers and lack of strong quantitation. Genome wide association studies have linked multiple match genes with AMD16 . Match activation prospects to anaphylatoxin production, leukocyte recruitment, and resultant NS-018 inflammation. In match receptor deficient mice, laser injury reduces mononuclear phagocyte recruitment and laser-induced choroidal neovascularization (CNV) area17 . Similarly, the C-C motif chemokine receptor 2 (CCR2) knockout mouse, which is usually deficient in monocyte recruitment to the tissue, demonstrates both decreased mononuclear phagocyte recruitment and laser-induced CNV area18 . These data link match and mononuclear phagocytes with eNOS experimental CNV and possibly neovascular AMD. In support of this association, match receptors are dysregulated on peripheral blood monocytes in patients NS-018 with neovascular AMD19,20 . These data demonstrate a strong association between AMD and mononuclear phagocytes. In this manuscript, we will use the experimental laser induced CNV model to characterize the mononuclear phagocyte populations in the mouse vision using multi-parameter circulation cytometry. Laser-induced CNV is the standard mouse model NS-018 of neovascular AMD, which exhibited the efficacy of current first collection neovascular AMD therapy21 . This protocol will describe enucleation of mouse eyes, ocular dissection, digestion into a single cell suspension, antibody staining, determination of.