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.

All procedures were approved by the Northwestern University Institutional Animal Care and Use Committee. C57BL/6 mice were group housed at a barrier facility at the Center for Comparative Medicine at Northwestern University (Chicago, IL). All animals were housed in 12/12 h light/dark cycle with free access to food and water.
1. Collection of ocular tissue
<ol>
	<li>Sacrifice 10-12-week-old C57BL/6J mice of either sex using regulated administration of carbon dioxide until mouse is unresponsiv...

<ol>
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W., Trautwein, C., Tacke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+monocytes+and+macrophages+for+the+inflammatory+response+in+acute+liver+injury.">Functional role of monocytes and macrophages for the inflammatory response in acute liver injury.</a> Frontiers in Physiology. 3, 56 (2012).</li><li>Gautier, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-expression+profiles+and+transcriptional+regulatory+pathways+that+underlie+the+identity+and+diversity+of+mouse+tissue+macrophages.">Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.</a> Nature Immunology. 13 (11), 1118-1128 (2012).</li><li>Sutti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1+Mediates+the+Development+of+Monocyte-Derived+Dendritic+Cells+during+Hepatic+Inflammation.">CX3CR1 Mediates the Development of Monocyte-Derived Dendritic Cells during Hepatic Inflammation.</a> Cells. 8 (9), 1099 (2019).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+Cells+Expressing+VEGF+and+Arginase-1+Following+Uptake+of+Damaged+Retinal+Pigment+Epithelium+Suggests+Potential+Mechanism+That+Drives+the+Onset+of+Choroidal+Angiogenesis+in+Mice.">Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice.</a> PLoS One. 8 (8), 72935 (2013).</li><li>Satpathy, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zbtb46+expression+distinguishes+classical+dendritic+cells+and+their+committed+progenitors+from+other+immune+lineages.">Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages.</a> The Journal of Experimental Medicine. 209 (6), 1135-1152 (2012).</li><li>Krause, T. A., Alex, A. F., Engel, D. R., Kurts, C., Eter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-Production+by+CCR2-Dependent+Macrophages+Contributes+to+Laser-Induced+Choroidal+Neovascularization.">VEGF-Production by CCR2-Dependent Macrophages Contributes to Laser-Induced Choroidal Neovascularization.</a> PLoS One. 9 (4), 94313 (2014).</li><li>Lin, W., Liu, T., Wang, B., Bi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ocular+dendritic+cells+in+uveitis.">The role of ocular dendritic cells in uveitis.</a> Immunology Letters. 209, 4-10 (2019).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nature Immunology. 17 (1), 34-40 (2016).</li><li>O'Koren, E. G., Mathew, R., Saban, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+reveals+that+microglia+and+recruited+monocyte-derived+macrophages+are+definitively+distinguishable+by+phenotype+in+the+retina.">Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by phenotype in the retina.</a> Science Reports. 6, 20636 (2016).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proceedings of the National Academy of Sciences U.S.A. 113 (12), 1738-1746 (2016).</li><li>M&#252;ller, A., Brandenburg, S., Turkowski, K., M&#252;ller, S., Vajkoczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+microglia,+and+not+peripheral+macrophages,+are+the+main+source+of+brain+tumor+mononuclear+cells.">Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.</a> International Journal of Cancer. 137 (2), 278-288 (2014).</li><li>McHarg, S., Clark, S. J., Day, A. J., Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+and+the+role+of+the+complement+system.">Age-related macular degeneration and the role of the complement system.</a> Molecular Immunology. 67 (1), 43-50 (2015).</li><li>Nozaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drusen+complement+components+C3a+and+C5a+promote+choroidal+neovascularization.">Drusen complement components C3a and C5a promote choroidal neovascularization.</a> Proceedings of the National Academy of Sciences U.S.A. 103 (7), 2328-2333 (2006).</li><li>Tsutsumi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+role+of+ocular-infiltrating+macrophages+in+the+development+of+choroidal+neovascularization.">The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization.</a> Journal of Leukocyte Biology. 74 (1), 25-32 (2003).</li><li>Subhi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+CD11b++Monocytes+and+Anti&#8211;Vascular+Endothelial+Growth+Factor+Injections+in+Treatment+of+Neovascular+Age-Related+Macular+Degeneration+and+Polypoidal+Choroidal+Vasculopathy.">Association of CD11b +Monocytes and Anti&#8211;Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.</a> JAMA Ophthalmology. 137 (5), 515-518 (2019).</li><li>Singh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Expression+of+CD46+and+CD59+on+Leukocytes+in+Neovascular+Age-Related+Macular+Degeneration.">Altered Expression of CD46 and CD59 on Leukocytes in Neovascular Age-Related Macular Degeneration.</a> AJOPHT. 154 (1), 193-199 (2012).</li><li>Kwak, N., Okamoto, N., Wood, J. M., Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+is+major+stimulator+in+model+of+choroidal+neovascularization.">VEGF is major stimulator in model of choroidal neovascularization.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3158-3164 (2000).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> Journal of Visualized Experiments. (106), e53502 (2015).</li><li>Mahajan, V. B., Skeie, J. M., Assefnia, A. H., Mahajan, M., Tsang, S. 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Multi-parameter flow cytometry allows for the quantitative analysis of multiple cell types in a complex tissue. In this report, we describe the flow cytometric detection of mononuclear phagocyte populations, including monocytes, dendritic cells, and macrophage subsets, after laser injury in the mouse eye. Liyanage et al recently reported a similar gating strategy to detect neutrophils, eosinophils, lymphocytes, DCs, macrophages, infiltrating macrophages, and monocytes27. Our gating strategy primar...

The innate immune system includes multiple cell types that stimulate complement 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 flow cytometric analysis in a mouse model of neovascular AMD2. This protocol is adaptable for mouse models of diabetic retinopathy and/or uveitis, but more extensive 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 the 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 described in the iris, choroid, ciliary body, and optic nerve in healthy eyes3. Alternatively, dendritic cell identification is more difficult; the most specific method of dendritic cell identification requires fate mapping using the Zbtb46-GFP reporter mouse9. Independent 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 identified in cornea, conjunctiva, iris, and choroid in normal eyes11. Microglia are specialized macrophages located in the retina, protected 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 available as a flow cytometry antibody14. Upon microglia activation, however, CD45 can be up-regulated15 and 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+Ly6ChighCX3CR1low expression, and non-classical monocytes demonstrate CCR2-Ly6ClowCX3CR1high markers5.
Due to the necessity of the quantitative analysis of marker expression, i.e., high versus low/dim levels, multi-parameter flow 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 flow cytometry is the lack of tissue architecture. This can be overcome by the ophthalmic dissection into the various 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 robust quantitation.
Genome wide association studies have linked multiple complement genes with AMD16. Complement activation leads to anaphylatoxin production, leukocyte recruitment, and resultant inflammation. In complement 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 deficient in monocyte recruitment to the tissue, demonstrates both decreased mononuclear phagocyte recruitment and laser-induced CNV area18. These data link complement and mononuclear phagocytes with experimental CNV and possibly neovascular AMD. In support of this association, complement receptors are dysregulated on peripheral blood monocytes in patients 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 eye using multi-parameter flow cytometry. Laser-induced CNV is the standard mouse model of neovascular AMD, which demonstrated the efficacy of current first line neovascular AMD therapy21. This protocol will describe enucleation of mouse eyes, ocular dissection, digestion into a single cell suspension, antibody staining, determination of laser voltages using single color controls, and gating strategy using fluorescence minus one (FMO) controls. For a detailed description of the laser-induced CNV model, please see a previous publication22. Using this protocol, we will define microglia, monocytes, dendritic cells, and macrophage populations. Furthermore, we will use MHCII and CD11c to further define macrophage subsets within the laser induced CNV model.

<table>
	<tbody>
		<tr>
			<td>0.6 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml microcentrifuge tubes</td>
			<td>Costar</td>
			<td>3620</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431031</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conicals</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS, 500 ml</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Henke Sass Wolf</td>
			<td>7886-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431032</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>Exel</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>3ml luer lock syringe</td>
			<td>Fisher Scientific</td>
			<td>14955457</td>
			<td></td>
		</tr>
		<tr>
			<td>41 x 41 x 8 mm polystyrene weigh dish</td>
			<td>Fisher Scientific</td>
			<td>08-732-112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conicals</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well, U-bottom assay plate without lid</td>
			<td>Falcon</td>
			<td>353910</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine reactive beads</td>
			<td>ThermoFisher</td>
			<td>A10628</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>FlowJo</td>
			<td>v 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat and anti-hamster Ig kappa bead set</td>
			<td>BD Biosciences</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation beads</td>
			<td>ThermoFisher</td>
			<td>01-1111-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Count beads</td>
			<td>ThermoFisher</td>
			<td>01-1234-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion enzyme</td>
			<td>Sigma Aldrich</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>National Optical and Scientific Instruments, Inc.</td>
			<td>DC3-420T</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-937</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block, rat anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Integra Miltex</td>
			<td>17035X</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamster anti-mouse CD11c BV421</td>
			<td>BD Biosciences</td>
			<td>562782</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>07-210-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead dye</td>
			<td>ThermoFisher</td>
			<td>65-0866-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution, 10x solution</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro titer tube and rack</td>
			<td>Fisher Scientific</td>
			<td>02-681-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse CD64 PE</td>
			<td>BioLegend</td>
			<td>139304</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse NK1.1 PECF594</td>
			<td>BD Biosciences</td>
			<td>562864</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2404</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipettor</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipet tips</td>
			<td>eppendorf</td>
			<td>22491806</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipettor</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipettor</td>
			<td>Gilson</td>
			<td>FA10003M</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2401</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipettor</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet man</td>
			<td>Fisher Scientific</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse B220 PECF594</td>
			<td>BD Biosciences</td>
			<td>562313</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD11b APC-Cy7</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 AlexaFluor 700</td>
			<td>BD Biosciences</td>
			<td>557958</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 PE</td>
			<td>BD Biosciences</td>
			<td>553786</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD4 PECF594</td>
			<td>BD Biosciences</td>
			<td>562314</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 FITC</td>
			<td>ThermoFisher</td>
			<td>11-0451-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>552848</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD8 PECF594</td>
			<td>BD Biosciences</td>
			<td>562315</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6C</td>
			<td>BD Biosciences</td>
			<td>561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6G PECF594</td>
			<td>BD Biosciences</td>
			<td>562700</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse MHC II AlexaFluor 700</td>
			<td>BioLegend</td>
			<td>107622</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Siglec F PECF594</td>
			<td>BD Biosciences</td>
			<td>562757</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Tim4 AlexaFluor647</td>
			<td>BD Biosciences</td>
			<td>564178</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Labnet</td>
			<td>311DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer, 40 mm nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water, 500 ml</td>
			<td>Gibco</td>
			<td>A12873-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge</td>
			<td>eppendorf</td>
			<td>5910 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

digestion of whole mouse eyes for multi-parameter flow cytometric analysis of mononuclear phagocytes

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.

Figure 1 showed uncompensated frequency histograms for the SCCs and cells for the red laser: Alexa647, Alexa700, and APC-Cy7. In Figure 1A, the magenta line depicted the peak of the SCC for Alexa647, while the cyan line showed&#160;the intended 0.5 Log difference. Notice that the peak in Alexa700 and APC-Cy7 was to the left (less bright) of the cyan line. Also note that the cells were all to the left of the magenta line. Any cells to the right of the SCC peak we...

Watch this Scientific Journal Video about Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes at JoVE.com

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

This protocol is designed to digest whole mouse eyes into a single cell suspension for multi-parameter flow cytometry of mononuclear phagocytes which include macrophages, monocytes, microglia, and dendritic cells. The main advantage of this technique is the ability to use quantitative analysis with fluorophore intensity to differentiate closely-related cell types using cell surface marker expression. Our protocol is optimized for the analysis of macrophage heterogeneity, including both resident and infiltrating populations in the laser-induced choroidal neovascularization model.However, these concepts and techniques can be equally applied to other models like diabetic retinopathy, experimental autoimmune uveitis, as well as manifestations of lupus. When attempting this protocol, understanding your cytometer's configuration is a critical starting point as all cytometers are not configured the exact same way. Thus, volumes of fluorescently conjugated antibodies may need to be titrated and/or fluorophores may need to be adjusted to optimize the protocol based on your cytometer's configuration.Begin by preparing the digestion buffer according to manuscript directions. Using a dissection microscope and 10X total magnification, remove the remaining conjunctiva and optic nerve from the eyes. Move clean eyes to a dry dissecting dish or a small weigh boat, then make two perforating wounds with a 90 degree angle between them through the visual axis.Use a syringe with a 30 gauge needle to inject 0.15 to 0.2 milliliters of digestion buffer into the vitreous cavity of each eye. Mince the eyes with spring scissors and fine forceps, then dissociate the lens tissue with blunt mechanical disruption to prevent clogging pipette tips in subsequent steps. Rinse the forceps and scissors with 0.75 milliliters of digestion buffer and replace the dish for each sample.Use a P1000 pipette to transfer the contents of the dish to a dissociation tube on ice, taking care to not lose any material. Rinse the dish with an additional 1.5 milliliters of digestion buffer and add it to the dissociation tube, bringing the total volume to between 3.25 and 3.5 milliliters. Dissociate the tissue on an electronic dissociator as described in the text manuscript, making sure that all tissue is at the bottom of the inverted dissociation tube and in contact with the digestion buffer.Then incubate the dissociation tube at 37 degrees Celsius and 200 RPM for 30 minutes. Repeat the tissue dissociation two more times, incubating the sample at 37 degrees Celsius and 200 RPM for 30 minutes in between the dissociations. To stop the reaction, add 10 milliliters of cold flow buffer and place the sample tubes on ice.To create a single cell suspension, strain the halted eye digestion reaction through a 40 micrometer filter placed on the top of a 50 milliliter conical centrifuge tube. Use the plunger from a 2.5 milliliter syringe to push any remaining pieces of undigested eye tissue through the filter. Wash the dissociation tube with 10 milliliters of flow buffer and rinse the filter.Then use the same plunger to again pass the undigested eye tissue pieces through the filter. Repeat this process with five milliliters of flow buffer. Then wash the dissociation tube and filter with a final 10 milliliters of flow buffer.Centrifuge the conical tube at 400 times G for 10 minutes at four degrees Celsius and decant the supernatant without disturbing the cell pellet. Break up the pellet by flicking the tube against another tube. Then add one milliliter of lysing solution and swirl the tube at room temperature for 30 seconds to lyse the red blood cells.Stop the reaction with 20 milliliters of flow buffer and centrifuge of the conical tube at 400 times G for 10 minutes at four degrees Celsius. Decant the supernatant without disturbing the cell pellet. Flick the tube against another tube.Then add five milliliters of cold HBSS. Repeat the centrifugation and remove the supernatant. Add 0.5 milliliters of live dead stain to each sample using a P1000 pipette, making sure to dissociate the pellet completely.Transfer the samples to 1.2 milliliter microtiter tubes and incubate them for 15 minutes in the dark. Meanwhile, count a 10 microliter aliquot of the sample with trypan blue and a hemocytometer. After the incubation, wash each sample by adding 400 microliters of cold flow buffer.Centrifuge the tubes in a microtiter tube rack at 400 times G for 10 minutes at four degrees Celsius and aspirate the supernatant without disturbing the cell pellet. Resuspend the cells completely in 500 microliters of cold flow buffer. Then repeat the centrifugation and remove the supernatant.Block up to five million living cells in 50 microliters of FC block. Completely dissociate the cell pellet and incubate it at four degrees Celsius for 20 minutes. Then add 50 microliters of antibody staining solution to each sample.Mix it completely and incubate it for another 30 minutes at four degrees Celsius. Forward scatter area versus side scatter area properties for all analyzed events from two eyes of a single mouse are shown here. Bead counts were cleaned and confirmed by plotting PE over APC-Cy7, creating a tight cluster of PE positive and APC-Cy7 negative events.Next, singlets in live cells were identified from all events. Singlets were positively correlated in forward scatter height versus forward scatter area while doublet and triplet cells had greater forward scatter area than forward scatter height. Live cells are forward scatter area positive and live dead stain negative.The initial gating strategy for the delineation of mononuclear phagocytes from live singlet cells is shown here. Live singlets were visualized using a CD45 versus CD11b plot. The absence of CD45 positive cells in the CD45 FMO confirmed the gate selection.Next, neutrophils, eosinophils, B cells, and K cells, and T cells were excluded by plotting CD45 positive live singlets with lineage gate versus CD11b. CD45-dim cells were differentiated from CD45-high cells. Macrophage subsets were also identified.Microglia were delineated in the CD45-dim cells with CD64 positive MHC2 low staining. MHC2 negative macrophages were identified as CD64 positive MHC2 negative. CD11c negative and CD11c positive macrophages were demonstrated as CD64 positive CD11c negative.and CD64 positive CD11c positive in the MHC2 positive cells. It was found that laser treatment increased the amount of MHC2 negative, CD11c negative, and CD11c positive macrophages. Dendritic cell counts were also upregulated by laser treatment while microglia and monocyte counts were not affected.Systemic perfusion had no effect on macrophage numbers. Following this procedure, florescence activated cell sorting rather than analysis alone allows for characterization of macrophage heterogeneity and its impact upon function via transcriptomic or proteomic studies.

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JoVE Journal

Immunology and Infection

4.8K visualizações.  Northwestern University. This protocol is designed to digest whole mouse eyes into a single cell suspension for multi-parameter flow cytometry of mononuclear phagocytes which include macrophages, monocytes, microglia, and dendritic cells. The main advantage of this technique is the ability to use quantitative analysis with fluorophore intensity to differentiate closely-related cell types using cell surface marker expression. Our protocol is optimized for the analysis of macrophage heterogeneity, including both reside...