Salome Murinello is supported by American Diabetes Association grant #1-16-PDF-072. This work was supported by grants to Martin Friedlander from the National Institutes of Health (National Eye Institute EY11254 and EY22025) and the Lowy Medical Research Institute.

All animals were treated in accordance with the ethical guidelines established by the Scripps Research Institute.
1. Preparation of Materials for Injection
<ol>
	<li>Sterilize a 33 G needle and syringe: disassemble and autoclave at 115&#160; &#959;C. Prepare needles for injection by rising in sterile phosphate buffered saline (PBS).</li>
	<li>Thaw fluorescently labeled particles at room temperature for 5 - 10 min. Prepare particle solution for injection by reconstituting to a 50 mg/ml solution in sterile PBS with Ca2+/Mg2+. 
	NOTE: AF488-labeled particles of fungal origin that have been validated for phagocytosis assays are used in this protocol21-23. For optimal uptake, prepare particles fresh and immediately before injection. 
	Note 2: Particle concentrations may be optimized for each specific experiment.</li>
</ol>
2. Intravitreal Injection of Bead Solution
NOTE: Two people are required to perform the injection, in a way such that the person performing the injection can hold the mouse and maintain the focus on the eyeball, while the other person passes the loaded syringe and pushes the plunger.
<ol>
	<li>Anaesthetize the rodent by intraperitoneal injection of 100 mg/ml ketamine and 10 mg/ml xylazine at a dose of 20 &#181;l/10 g of body weight. Before injection, use toe pinch to assess level of anesthesia. 
	NOTE: Isoflurane has a profound effect on myeloid cell function, thus, its use throughout this assay should be avoided24,25.</li>
	<li>Load needle with 0.5 &#181;l of fluorescently labeled particle solution.</li>
	<li>Lay the mouse sideways under a surgical microscope (Figure 1A-B). Use a soft material, e.g., a gel pack, for better positioning of the mouse. For young mice in which the eyes are not yet open, gently open the eyelids with the help of fine 45o angled forceps by creating a fissure along the slit from which the eyelids will eventually open.
	<ol>
		<li>With the help of fine 45o angled forceps carefully apply pressure around the eyelid, so that the eyeball pops slightly out of the socket. Hold the head with two fingers just above the ear and by its jaw and gently stretch the skin in parallel to the eyelids to keep the eye slightly out of the socket. Be careful not to grasp too close to the throat (Figure 1B).</li>
	</ol>
	</li>
	<li>To puncture the eyeball, insert the syringe&#39;s needle in the corneal limbus (where the cornea and sclera connect). This is visible as a gray circle in pigmented mice. Retract the needle slightly to expel a small volume of vitreous fluid and then inject. The person performing the injection should gently hold the mouse with one hand and the needle with the other; the second person should slowly push the plunger.</li>
	<li>Retract the syringe slowly. Apply eye moisturizing drops to keep the eye hydrated.</li>
	<li>Let the rodent recover in a cage over a heat pad and continue to monitor the animal. If working with pups, do not return the animal to a cage with other alert animals until it is breathing and capable of spontaneous movement. If working with adults, do not return the rodent to a cage with other alert animals until it regains sternal recumbence.</li>
</ol>
3. Harvesting of Retinal Tissue
NOTE: Retinal tissue from eyes not injected with fluorescently labeled particles should be collected as a control for flow cytometric analysis. Though the assay can be performed using a single retina, for best performance, two retinas should be pooled together.
<ol>
	<li>Collect retinal tissue 3 hr after intravitreal injection of fluorescently labeled particles. 
	NOTE: While time after injection of the particle solution may be optimized for each specific experiment, we found that 3 hr after injection, particle uptake could be seen throughout most layers of the retina (Figure 1C-D).</li>
	<li>Sacrifice mice by cervical dislocation.</li>
	<li>Collect the eyeballs by gently pressing against the eyelid with the help of fine 45o angled forceps to proptose the eyeball. Position the forceps behind the eyeball and pull.</li>
	<li>Dissect the retina under a dissecting microscope. Transfer the eyeball to a Petri dish containing a small amount of PBS with Ca2+/Mg2+. In a dry area of the Petri dish, perforate the eyeball in the corneal limbus with the tip of superfine forceps.</li>
	<li>Hold the eyeball with the help of fine 45o angled forceps and use spring scissors to cut around the corneal limbus, until roughly half of the corneal limbus circumference is cut.</li>
	<li>Hold the eyeball with the fine 45o angled forceps and bring the eyeball into PBS. With a second pair of fine 45o angled forceps tear the cornea and sclera apart. The lens and retina will come out intact.</li>
	<li>Separate the lens and retina. Collect the retina and transfer to a 5.4 ml polystyrene test tube containing 2 ml of PBS with Ca2+/Mg2+.</li>
</ol>
4. Preparing a Single Cell Suspension
<ol>
	<li>Prepare single cell suspensions using a neuronal tissue dissociation kit with minor modifications to the manufacturer&#39;s instructions. Briefly, triturate retinas by pipetting up and down with a P1000 pipette and performing an enzymatic digestion at 37 oC without shaking.</li>
</ol>
5. Staining Single Cell Suspensions for Flow Cytometric Analysis
<ol>
	<li>Resuspend cells in 200 &#181;l of staining buffer (Dulbecco&#39;s phosphate-buffered saline with 0.2% bovine serum albumin (BSA) and 0.09% sodium azide) and transfer to a U-bottom 96-well plate. Centrifuge for 5 min at 130 x g. 
	NOTE: Sodium azide is harmful to humans and the environment. Use appropriate personal protective equipment and discard waste in accordance with local regulations.</li>
	<li>Invert the plate over a sink to discard supernatant. To block Fc receptors, resuspend cells in 25 &#181;l of stain buffer containing 5 &#181;g/ml of anti-mouse CD16/CD32 antibody per well. Incubate for 5 min at room temperature.</li>
	<li>Add 25 &#181;l of staining buffer containing 0.5 &#181;g/ml of anti-mouse Ly6C-APC-Cy7, 0.5 &#181;g/ml of anti-mouse Ly6G-Pe-Cy7 and 2.5 &#181;g/ml of anti-mouse CD11b-AF650. Incubate for 15 min at room temperature in the dark.</li>
	<li>Centrifuge for 5 min at 130 x g. Invert the plate to discard supernatant. Wash by resuspending cells in 200 &#181;l of staining buffer.</li>
	<li>Centrifuge for 5 min at 130 x g. Resuspend in 200 &#181;l of stain buffer containing 0.5 &#181;g/ml of propidium iodide (PI). Transfer to 1.2 ml microtiter tubes.</li>
	<li>Wash wells with an additional 100 &#181;l of staining buffer containing 0.5 &#181;g/ml of PI and pool with the previous 200 &#181;l in the 1.2 ml microtiter tubes. A total volume of 300 &#181;l of stained cells is obtained.</li>
</ol>
6. Flow Cytometric Analysis
<ol>
	<li>Using a conventional three laser (violet, blue, and red laser), avoid PE, PerCP-Cy5.5, BV510, and PI dyes due to significant spillover from the extremely bright fluorescent AF488-particles. Use a fourth (yellow) laser to optimize this assay, as it allows for a PE-labeled antibodies and PI (in the mCherry channel instead of the PerCP-Cy5.5 channel) to be used (Figure 2). 
	NOTE: If a yellow laser is not available, use a dead cell exclusion dye in another channel.</li>
	<li>Gate on PI negative cells (PI-) to exclude dead cells (Figure 2B).</li>
	<li>After excluding dead cells, gate on CD11b positive cells (CD11b+), this will include all myeloid cells (Figure 2C).</li>
	<li>Within the CD11b+ population, gate on Ly6C-/Ly6G- to exclude neutrophils (CD11b+/Ly6G+) and monocytes (CD11b+/Ly6C+; Figure 2D). After gating on microglia (here defined as CD11b+/Ly6C-/Ly6G- for simplicity), two clear populations should be visible; one negative and one positive for the fluorescently labeled particles. Phagocytic cells will have taken up particles and are therefore AF488+ (Figure 2E). 
	NOTE: Ly6C positive or Ly6G positive populations can also be analyzed to measure particle uptake using this staining approach. The percentage of phagocytic CD11b+ cells correlates well with the percentage of phagocytic microglia (Figure 2F). For researchers without flow cytometry experience, a simpler analysis with CD11b staining alone can be performed, albeit this will include phagocytic neutrophils and monocytes, as well as microglia. Within this CD11b+/Ly6C-/Ly6G- population, these cells are &#62; 99% microglia as judged by staining with the markers F4/80 and CX3CR1; these markers can be added but are not necessary in our hands.</li>
</ol>

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The authors have nothing to disclose.

There are three critical steps in this method: (1) intravitreal injection of fluorescently labeled particles; (2) harvesting and preparation of retinal tissue; and (3) flow cytometric analysis. We recommend that researchers practice intravitreal injections prior to performing the method we present here. Albino mice (e.g., BALB/c) and a colored solution (e.g., fluorescently labeled particles) can be used for easy visualization of the needle and injected solution. Intravitreal injections are challenging and if not performed correctly will lead to biased and variable results. Common problems associated with poor injection technique are perforation of the lens and/or damage to the retina, which may result in bleeding and inflammation. To minimize the risk of trauma to the eye, the rodent should be in a stable position with minimal head movement. To penetrate the eyeball, the syringe should be inserted slowly and not pushed too far to avoid hitting the lens. Another common complication is reflux of the injected material out of the eye. To avoid this, the plunger should be depressed slowly and, after injection, the eyeball should be allowed to retract into the eye socket and the syringe should be retracted slowly. The best intravitreal injections activate microglia at low levels, but clear differences between untreated and treated groups can be measured. It is essential that appropriate controls (e.g., non-challenged mice) are used in each experiment to establish baseline phagocytic levels. Another aspect of this method that requires practice is the retinal dissection. To ensure comparable single cell preparations across specimens, retinal tissue must be collected swiftly and with minimal possible tissue disruption. Performing the last steps of dissection in PBS (see 3.4 - 3.7) will facilitate collection. Finally, it is important to prepare the retinal tissue for flow cytometry correctly. Activated microglia upregulate Fc receptors and thus nonspecific binding to the Fc portion of antibodies may occur7. An Fc block must be performed to prevent nonspecific staining. Selection of appropriate fluorophores and compensation set up is also important27.
We routinely use mice aged 10 to 20 postnatal days for these experiments. However, this protocol can also be used to assess microglial phagocytic function in adult mice (Figure 3C). Of note, due to presence of the hyaloid vasculature, populations of exogenous myeloid cells (Ly6C+ or Ly6G+) are more prominent in young mice (approximately 40 - 50% of all CD11b+ cells) than in adults (approximately 5% of all CD11b+ cells). Researchers should determine the age most appropriate for their experiments. The protocol presented here allows for robust detection of the levels of phagocytic microglia in the retina. However, we recommend that three to four biological replicates be used in each experiment and that at least three independent experiments are performed. While the particles used in this protocol have been validated for phagocytic assays21-23, if additional validation is required, immunohistochemical co-localization of the particles with specific phagocytic markers could be performed.
One potential limitation of this method is differential access of microglia to the particles. In the normal retina, microglia reside in two layers, the deeper outer plexiform layer, and the more superficial inner plexiform layer, which is closer to the vitreous7. Upon stimulation, or during aging, microglia can migrate throughout all retinal layers. In this method, fluorescently labeled particles are injected into the vitreous. Three hours after injection this results in particle accumulation throughout the retina and particularly along the vitreal surface. Researchers may further optimize the concentration of particles or time after injection for their experiments. It is likely that microglia in closer proximity to the vitreous will have easier access to the particles. Nevertheless, we show that the particles spread across the retina and are taken up by microglia across all layers but not in retinal pigmented epithelium (RPE) cells, probably due to limited bead access (Figure 1C-E). We have yet to determine whether subretinal microglia (which can accumulate with age) would gain access to the particles. If this is important to the researcher, this protocol could be performed after careful subretinal injection of the particles. Another important consideration is that there may be inherently different behaviors between retinal microglia and microglia in other regions of the CNS. It is well known that specific brain regions have different susceptibilities to disease. For example, in Parkinson&#39;s disease, neurons in the substantia nigra are mostly affected, while in Alzheimer&#39;s disease, the hippocampal neurons are mostly affected28. Thus, when studying the pathology of a specific disease, brain region is likely to be an important factor. A similar protocol to the one described here could be performed after intracranial injection to explore potential differences in the phagocytic response between retinal and brain microglia. The retina has many common features with other regions of the brain, including the presence of a blood-retina-barrier20. Retina microglia and brain microglia originate from the same yolk-sac progenitors and appear very similar with regard to morphology and cell-surface marker expression7,29. Furthermore, several brain diseases (e.g., Alzheimer&#39;s disease, stroke, and multiple sclerosis) have ocular manifestations, indicating that the retina is also affected20. While researchers should be aware of potential regional differences, we believe this is an informative model for all microglia research, and suggest that all neuroscientists studying microglial phagocytosis could use this protocol.
A distinct advantage to performing this assay in the eye is that multiple techniques have been developed to induce diverse neurogenic stresses in the retina, and the stress responses can be monitored in vivo and quantified using standardized protocols30. By inducing neurogenic stress and then performing the assay described here, researchers can analyze microglia function across a broad spectrum of insults that vary in severity. Finally, when combined with flow cytometry sorting techniques, this protocol has the advantage over immunohistochemistry that it may allow for in depth analysis of phagocytic microglia (e.g., qPCR, cell-culture, proteomics).
Uptake of particles has previously been used to measure phagocytic function, both in in vitro and in vivo systems. The latter involved intracranial injection of particles, harvesting and freezing of brain tissue, cryosectioning, immunostaining, imaging and post-imaging analysis18. The protocol presented here allows for much faster quantification of retinal microglia phagocytic function. It has the advantage over in vitro systems that the highly sensitive microglia are not removed from their microenvironment, allowing for assessment of phagocytic function in a physiological context. Specifically, it should enable the testing of how defects in other cell types (e.g., genetic ablation of genes in neurons) affect microglia phagocytic function. It also has the advantage over previous in vivo assays that it uses flow cytometry, allowing for fast, quantitative, and accurate detection of phagocytic cell populations.

The overall goal of this method is to accurately assess and quantify in vivo microglial phagocytosis. Microglia are the tissue resident macrophages of the central nervous system (CNS). They perform a variety of functions to ensure maintenance of tissue homeostasis. These include immune surveillance, secretion of neurotrophic factors and, of pivotal importance, phagocytosis1. Microglial phagocytosis is key in several important events during development of the brain and retina, such as phagocytosis of irrelevant synapses (synaptic pruning) and removal of apoptotic neurons2-4. Furthermore, microglial phagocytosis of damaged or apoptotic neurons, cellular debris, and invading microbes has been shown to be essential for maintaining CNS homeostasis through adulthood5. Finally, microglial phagocytosis has been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer&#39;s disease and age-related macular degeneration, where it has been suggested that defective or insufficient phagocytic capacity may contribute to the build-up of amyloid-&#946; (A&#946;) plaques and drusen, respectively6,7.
Microglial function is tightly regulated by their microenvironment, notably by soluble factors such as tumor-growth factor &#946; or cell-cell interactions. Neurons constitutively express several cell surface ligands, such as CD200 and CX3CL1, while microglia exclusively express the respective receptors CD200R and CX3CR1. These receptors contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their intracellular portion. These inhibitor receptors are critical for preventing the over-stimulation of microglia, which can contribute to neuroinflammation. Thus, under normal physiological conditions, cell-cell interactions between neurons and microglia keep microglia in a quiescent state. During tissue injury, however, neurons can down-regulate expression of these ligands, removing their inhibitory effect on microglia activation. Microglial function (including phagocytosis) is thus tightly linked to their microenvironment8. Nevertheless, to date, there are no standardized assays to study microglia phagocytosis in a physiological context or in a way that fully replicates their CNS microenvironment.
Several assays have been developed to measure phagocytic activity of microglia in vitro, where primary microglia or microglia cell lines are cultured with target cells (e.g., apoptotic neurons) or fluorescently labeled beads. Target uptake is then assessed using fluorescent imaging microscopy or flow cytometry9-12. These assays allow testing of how pharmacological or genetic manipulation may affect microglial phagocytosis and, while informative, fail to fully replicate the complex in vivo environment. Indirect methods for examining microglial phagocytosis in vivo have been reported: these are accomplished by staining of molecules thought to be involved in phagocytosis (e.g., CD68), assessing physical proximity of microglia and targets for phagocytosis (e.g., compromised neurons or synaptic elements), or by immunohistochemical detection of phagocytic targets within microglial cells (e.g., A&#946;)13-17. Two studies have used more direct approaches to assess microglia phagocytosis in vivo. Hughes and colleagues have used imaging techniques to measure microglial uptake of beads delivered via the intracranial route18. Sierra et al. developed a refined method to quantitatively assess microglia phagocytosis of apoptotic cells using complex imaging techniques4. However, these methods involve complicated protocols for tissue preparation, sectioning, imaging, and analysis. We have previously used flow cytometric analysis to assess phagocytosis of photoreceptor outer segments by retinal pigmented epithelium (RPE) cells in culture19. Here, we describe a protocol to rapidly assess uptake of fluorescently labeled particles by retinal microglia as a quantitative measure of in vivo microglia phagocytosis.
The protocol we describe here allows for reliable and quantitative measurement of retinal microglial phagocytosis in just under six hours in three critical steps: (1) intravitreal delivery of fluorescently labeled particles, (2) harvest and preparation of retinal tissue, and (3) flow cytometric analysis. The method we have developed is a robust method to assess microglial phagocytosis in the retina, and it can be successfully used to test how various compounds or genetic manipulation alter this key microglial function in physiological settings. As a specialized area of the CNS, the retina is an easily accessible model system to study microglia function20. While this method was developed in the eye, we believe it can be useful for all neuroscientists investigating microglia phagocytic function.

<table><tbody><tr><td>Stereomicroscope</td><td>Nikon</td><td>Discontinued</td><td></td></tr><tr><td>Hamilton&amp;nbsp;syringe, 600 series</td><td>Sigma</td><td>26702</td><td></td></tr><tr><td>33 gauge, Small Hub RN NDL, 0.5 in, point style 4 - 12o</td><td>Hamilton</td><td>7803-05</td><td></td></tr><tr><td>Zymosan A (&lt;em&gt;S. cerevisiae&lt;/em&gt;) BioParticles, Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific</td><td>Z-23373</td><td>Prepare immediately before injection</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>Need two</td></tr><tr><td>Dumont #5SF Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Vannas Spring Scissors - 3 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-10</td><td>Curved</td></tr><tr><td>Neural Tissue Dissociation Kit &amp;ndash; Postnatal Neurons</td><td>Miltenyi Biotec</td><td>130-094-802</td><td></td></tr><tr><td>5 ml Polystyrene Round-bottom Tube</td><td>Falcon</td><td>352054</td><td></td></tr><tr><td>96 well U-bottom plate</td><td>Falcon</td><td>353077</td><td></td></tr><tr><td>Stain Buffer (BSA)</td><td>BD Biosciences</td><td>554657</td><td></td></tr><tr><td>CD11b-BV650 Antibody</td><td>BioLegend</td><td>101259</td><td></td></tr><tr><td>Ly6C-APC-Cy7</td><td>BioLegend</td><td>128025</td><td></td></tr><tr><td>Ly6G-PE-Cy7</td><td>BioLegend</td><td>127617</td><td></td></tr><tr><td>Propidium Iodide</td><td>BD Biosciences</td><td>556463</td><td></td></tr><tr><td>Purified anti-mouse CD16/32 Antibody</td><td>BioLegend</td><td>101301</td><td></td></tr></tbody></table>

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.

Here we describe a method to rapidly and reliably quantify the number of phagocytic retinal microglia in a physiological setting using flow cytometric analysis (Figure 2). This method can be adapted to test the effect of compounds and/or genetic manipulation on the phagocytic capacity of microglia (Figures 3A, 3B). It can also be used in young (10 - 20 days postnatal) or adult mice (Figure 3C). Varying doses of lipopolysaccharide (LPS) were administered intraperitoneally. 24 hr after LPS challenge, the protocol described here was used to assess retinal microglia phagocytic function (Figure 3A). A dose of 1.42 mg/kg of LPS induced a statistically significant increase in the percentage of phagocytic microglia when compared to vehicle control (Figure 3B), as would be expected26.
<img alt="Figure 1" src="/files/ftp_upload/54677/54677fig1.jpg" /> 
Figure 1: Intravitreal Injection Setup and Representative Retinal Section showing Fluorescently Labeled Particles in Retinal Layers. (A) A gel pack is positioned under the dissection microscope. (B) A rodent is held as shown to position the eye for intravitreal injection. (C) Three hours after injection fluorescently labeled particles can be seen throughout most retinal layers. (D,E) CX3CR1GFP/GFP mice and particles labeled with a red fluorophore were used to visualize particle uptake by microglia. Microglia in the deeper (D) and superficial (E) layers take up particles. Scale bars - 20 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54677/54677fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54677/54677fig2.jpg" /> 
Figure 2: Gating Strategy to Select Phagocytic Microglia. (A) Single cells suspensions from retinal tissue from a p10 mouse are analyzed. (B) After excluding dead cells (PI+), (C) CD11b+ cells are selected. (D) To select only for microglia, CD11b+ neutrophils (Ly6G+) and monocytes (Ly6C+) are excluded. (E) Phagocytic microglia will have taken up fluorescently labeled particles. (F) The number of phagocytic CD11b+ cells is similar to that of microglia (CD11b+Ly6G-Ly6C-). n= 9, pooled from three independent experiments; error bars represent mean &#177; SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/54677/54677fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54677/54677fig3.jpg" /> 
Figure 3: Microglia Phagocytic Function can be Assessed after LPS Challenge and in Pups and Adult Mice. (A) Young mice (p10) were challenged with intraperitoneal LPS at varying doses, 24 hr before assessing phagocytosis. (B) Challenge with 1.42 mg/kg resulted in a significantly increased percentage of phagocytic retinal microglia when compared to vehicle controls (p= 0.0021). (C) This protocol can be used to measure retinal microglial phagocytic function in pup and adult mice alike. There is a significantly smaller percentage of phagocytic microglia in adult mice when compared to young (p10) mice (p= 0.019) not challenged with LPS. n= 9 -12, pooled from three to four independent experiment; error bars represent mean &#177; SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/54677/54677fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

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.

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, ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.5K Views.  The Scripps Research Institute. 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.

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Medicine

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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.

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. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Neuroscience

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Source: Murinello, S. et al., <a href="https://app.jove.com/v/54677">Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

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