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 unresponsive and no longer inhaling. Perform cervical dislocation or other approved secondary method to ensure euthanasia. 
	NOTE: Transcardial perfusion can be performed to remove circulating leukocytes that are not in the ocular tissues. In this experiment, systemic perfusion does not reduce the number of microglia, monocytes, macrophages, or dendritic cells detected in untreated or laser treated whole eyes. Therefore, the majority of these cell types are located in the ophthalmic tissues and this step is optional.</li>
	<li>To enucleate eyes, push down near the eye socket while placing a pair of curved forceps gently under the eye as it stands out of the socket. Pinch forceps closed under the eye without squeezing the actual eye. Dislodge eye from the surrounding conjunctiva (please see prior publication)23.</li>
	<li>Pull eye laterally until the optic nerve is the only remaining connection to detach the eye. The optic nerve often severs with tension, but a small scissor is sometimes necessary to cut the optic nerve. Place the eye in cold 1x Hank&#8217;s Buffered Saline Solution (HBSS) with calcium and magnesium in 1.7 mL microcentrifuge tube.</li>
</ol>
2. Digestion of ocular tissue
<ol>
	<li>Prepare the digestion buffer containing 0.1 mg/mL digestion enzyme and 0.2 mg/mL DNase in cold HBSS. Reconstitute 5 mg digestion enzyme in 2 mL of sterile water initially. Prepare an initial dilution of 10 mg/mL DNase in HBSS. For every 10 mL of the digestion buffer (sufficient for 3 animals) mix 9.4 mL of cold HBSS with 0.4 mL of reconstituted digestion enzyme and 0.2 mL of diluted DNase I. 
	NOTE: These concentrations were empirically determined over multiple experiments to provide optimal levels of antibody staining of single cells, while reducing cell death. Collagenase D was tried as an alternative to digestion enzyme with reduced cell viability and decreased CD45+ cells. Please see Discussion for more details regarding the iterative process to optimize digestion.</li>
	<li>Under a dissecting microscope at 10x total magnification, remove the remaining conjunctiva and optic nerve using fine forceps and spring scissors in cold HBSS.</li>
	<li>Move clean eyes to a dry dissecting dish or small weigh boat. Inject 0.15-0.20 mL/eye of digestion buffer via syringe fitted with 30 G needle into the vitreous cavity after two perforating wounds are made in the eye through the visual axis and 90&#730; away from this first wound for digestion buffer egress.</li>
	<li>Mince whole eyes using spring scissors and fine forceps with all pieces smaller than 0.5 mm x 0.5 mm. Dissociate lens tissue with blunt mechanical disruption via forceps to prevent clogging pipette tips in subsequent steps. For each sample, rinse forceps and scissors each with 0.75 mL of digestion buffer into small dish. Use a new dish for each sample.</li>
	<li>Transfer contents of the dish to a dissociation tube on ice using P1000 pipette taking care to not lose any material in the pipette transfer. Rinse the dish with additional 1.5 mL of digestion buffer bringing the total volume in dissociation tube to 3.25-3.50 mL.</li>
	<li>Place the dissociation tube inverted onto electronic dissociator and run cycle &#8220;m_brain_03_01&#8221;, consisting of 1 min unidirectional rotation at 200 rpm at room temperature, which is a pre-loaded program. Ensure all tissue is at the bottom of the dissociation tube in contact with digestion buffer and the rotor for this and all remaining steps.</li>
	<li>Place the dissociation tube in shaking incubator for 30 min at 200 rpm at 37 &#730;C.</li>
	<li>Repeat steps 2.6 and 2.7 one additional time. Following second 30 min incubation perform one final mechanical digestion using electronic dissociator as in step 2.6.</li>
	<li>Stop the reaction by adding 10 mL of cold flow buffer and place tubes on ice.</li>
</ol>
3. Preparation of cell suspension
<ol>
	<li>To create single-cell suspension, pour the halted eye digestion reaction through a 40 &#956;m filter placed on the top of a 50 mL conical centrifuge tube. Using the plunger from a 2.5 mL syringe, push any remaining pieces of undigested eye tissue through the filter.</li>
	<li>Wash the dissociation tube with 10 mL of Flow buffer and rinse filter before using the same plunger to again pass undigested eye tissue pieces through the filter.</li>
	<li>Repeat step 3.2 using 5 mL of Flow buffer.</li>
	<li>Wash the dissociation tube and filter with a final 10 mL of Flow buffer. 
	NOTE: Total volume of digestion and flow buffer is about 38-40 mL for each sample.</li>
	<li>Centrifuge the conical tube at 400 x g for 10 min at 4 &#730;C. Decant the supernatant without disturbing the cell pellet.</li>
	<li>Prepare 1x lysing solution by adding 10x lysing solution to sterile water. Break up the pellet by flicking tube against another tube. To lyse red blood cells, add 1 mL of 1x lysing solution for 30 s with swirling at room temperature (RT).</li>
	<li>Stop the reaction with 20 mL of Flow buffer.</li>
	<li>Centrifuge the conical tube at 400 x g for 10 min at 4 &#730;C. Decant the supernatant without disturbing the cell pellet.</li>
	<li>Dissociate the pellet by flicking tube against another tube. Add 5 mL of cold 1x HBSS per tube.</li>
	<li>Centrifuge tubes at 400 x g for 10 min at 4 &#730;C. Aspirate supernatant.</li>
</ol>
4. Staining of cell suspension for mononuclear phagocytes
<ol>
	<li>Prepare 0.5 mL of Live/Dead stain per sample by diluting Live/Dead dye 1:5,000 in cold HBSS.</li>
	<li>Add Live/Dead stain to each sample using a P1000 pipette making sure to dissociate the pellet completely. Transfer samples to 1.2 mL micro titer tubes.</li>
	<li>Take a small aliquot (10 &#956;L) to count cells using a hemocytometer or automatic cell counter (see 4.4). Incubate samples with Live/Dead stain for 15 min at room temperature in the dark.</li>
	<li>Count cells using Trypan blue to determine the number of live cells per sample. 
	NOTE: Typically, 2 eyes from a 10-12-week-old C57BL/6J mouse contain less than 2 x 106 living cells.</li>
	<li>Wash samples by adding 400 &#956;L of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 &#730;C. Aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend cells completely in 500 &#956;L of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 &#730;C. Aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Block up to 5 x 106 living cells in 50 &#181;L of Fc block (1:50 dilution in cold Flow buffer of purified rat anti-mouse CD16/CD32). If more than 5 x 106 living cells, increase volumes appropriately.</li>
	<li>Add Fc block to the pellet making sure to completely dissociate the sample. Incubate for 20 min at 4 &#730;C.</li>
	<li>Add 50 &#956;L of antibody staining solution (see Table 1 for choice and amount of each antibody that is included in the solution) per 5 x 106 living cells directly to cells in Fc block and mix completely. Incubate for 30 min at 4 &#730;C. 
	NOTE: Antibody quantities are dependent upon the flow cytometer configuration and should be titrated independently for every lab and instrument. See Table 2 for instrument configuration of filters and mirrors. To titrate antibodies, start with manufacturer&#8217;s recommendation, and calculate the staining index. Perform a test run of varying antibody concentrations (both above and below the manufacturer&#8217;s recommendation) and choose the lowest concentration of antibody where the staining index is maintained. Also, ensure that positive staining that is less bright than the single-color control. Use unstained cells to ensure that negatively stained cells are on scale and have a peak at or less than 1 x 102. Use a fluorescence minus one control to define the positively stained cells.</li>
	<li>Wash samples by adding 700 &#956;L of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 &#730;C. Aspirate the supernatant without disturbing the cell pellet.</li>
	<li>Optionally, after antibody staining, the samples can be fixed with 500 &#956;L of 2% paraformaldehyde in Flow buffer. Fix samples for 15 min at room temperature. Then, perform one wash as described in 4.10. Store fixed samples at 4 &#730;C. Count beads should be added on the day of flow cytometry data collection. Fixed samples should be run on flow cytometer in 1-2 days. 
	CAUTION: Paraformaldehyde is a corrosive and a health hazard.</li>
	<li>Wash samples again using 500 &#956;L of cold Flow buffer. Centrifuge tubes in a micro titer tube rack at 400 x g for 10 min at 4 &#730;C. Aspirate half of the supernatant without disturbing the cell pellet.</li>
	<li>Resuspend pellets and transfer to a 96 well, U-bottom assay plate. Centrifuge plate at 400 x g for 5 min at 4 &#730;C. To decant the supernatant, turn plate over and in one strong shake dislodge the liquid into a sink basin, without disturbing pellet.</li>
	<li>In a new 1.2 mL micro titer tube, place 50 &#956;L of well vortexed count beads.</li>
	<li>Resuspend pellets in 96 well plate in 200 &#956;L of cold Flow buffer. Add cell mixture on the top of beads from prior step. Run on flow cytometer in total volume of 250 &#956;L / micro titer tube.</li>
</ol>
5. Collection of flow cytometry data
<ol>
	<li>Turn on and prepare flow cytometer. Instructions listed here are for a modified four laser cytometer (Table 2).</li>
	<li>Run a test sample, which includes a small aliquot from each sample, to ensure that all cells are on scale for each detector. Adjust voltages so that all cells are on scale.</li>
	<li>Run single color controls (SCC) for every fluorophore used in staining cocktail (Table 3). Place micro titer tube into a larger 5 mL polystyrene tube for placement onto the flow cytometer stage. 
	NOTE: Root fluorophores like BV421 (Pacific Blue), FITC, PE, Alexa647 (APC), and Alexa700 do not require the same antibody as the cocktail (i.e., CD19 PE for CD64 PE). However, tandem fluorophores like PE-CF594, PE-Cy7, or APC-Cy7 require the same antibody for SCC as cocktail due to potential dissociation of the conjugated fluorophores.
	<ol>
		<li>Create the SCC for BV421 by adding 2 drops of compensation beads to a 1.2 mL titer tube along with 1 &#956;L of hamster anti-mouse CD11c BV421. Compensation beads are used because CD11c BV421 is an IgG lambda subtype rather than kappa as described in 5.3.3. Incubate for 15 mins at 4 &#730;C. Add 0.2 mL of flow buffer.</li>
		<li>Create the SCC for the Live/Dead dye by adding one drop of positive staining beads (green cap) from the amine reactive beads, followed by 1 &#956;L of Live/Dead dye. Incubate for 15 min at room temperature in the dark. Add one drop of the negative beads (white cap) from the amine reactive beads. Add 0.2 mL of flow buffer.</li>
		<li>Create the remaining SCCs by adding one drop of positive staining beads (green cap) from the anti-rat and anti-hamster Ig kappa bead set, followed by the antibody quantity listed (Table 3) in a 1.2 mL titer tube. Incubate for 15 min at 4 &#730;C. Add one drop of the negative beads (white cap) from the anti-rat and anti-hamster Ig kappa bead set. Add 0.2 mL of flow buffer.</li>
		<li>Vortex all bead mixtures completely. Store SCCs at 4 &#730;C for up to 3 days. 
		NOTE: The SCC for fluorescent reporter mice is unstained cells.</li>
		<li>When running SCCs, set voltages such that the SCC sample has a peak greater than any fluorescence for a sample of cells in the same detector channel. In addition, voltages should be set so that any SCC has at least a 0.5 Log difference between the histogram peak in the channel of interest versus any other channel (Figure 1).</li>
	</ol>
	</li>
	<li>Run samples on &#8220;Low&#8221; keeping events/second below 4000. If flow rate cannot be lowered appropriately, dilute samples with additional cold flow buffer. Record the volume added as this will be necessary for the count calculation (see representative results).</li>
	<li>Collect at least 3 x 106 events per sample. This may be higher than the cell number due to pigment granules and debris counting as events on your cytometer.</li>
	<li>Record a Fluorescence Minus One (FMO) for each fluorophore other than Live/Dead for proper gating strategy in section 6.4. An FMO is a sample that has been stained with all fluorophores except one.</li>
	<li>Clean and shut down flow cytometer according to manufacturer or facility instructions.</li>
</ol>
6. Annotation of flow cytometry data
<ol>
	<li>Open a new worksheet using analysis software. Import FCS files from cytometer for all samples and single-color controls.</li>
	<li>Use SCCs to create a compensation matrix.</li>
	<li>Apply the compensation matrix to your stained samples and FMOs.</li>
	<li>Use the FMOs to determine positive staining to draw gates.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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. ...

digestion-whole-mouse-eyes-for-multi-parameter-flow-cytometric

Research

JoVE Journal

Immunology and Infection

4.9K Views.  Northwestern University. 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.

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Medicine

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Neuroscience

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

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

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Przeglądaj więcej filmów

Mouse Eye Enucleation for Remote High-throughput Phenotyping

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells