We thank Wendy Salmon and Nicki Watson of the Keck facility at the Whitehead Institute of MIT for imaging.

1. Preparation of DC2.4 and RAW 264.7 Cell Lines
NOTE: The macrophage-like cell line RAW 264.7 and the dendritic cell line DC2.4 are both murine origin, and the following conditions were used to grow the cells.
<ol>
	<li>Grow RAW 264.7 cells in DMEM (Dulbecco&#39;s Minimal Eagle&#39;s medium) supplemented with 10% inactivated Fetal Bovine Serum (FBS) at 37 &#176;C in a humidified incubator with 5% CO2.</li>
	<li>Grow DC2.4 cells in similar conditions with that of RAW 264.7 cells, except use RPMI (Roswell Park Memorial Institute) medium supplemented with L-Glutamine instead of DMEM.</li>
</ol>
2. Preparation of Fluorescent Conjugated Particulates: C. albicans, Zymosan, IgG-coated Beads
<ol>
	<li>Growing Candida albicans
	<ol>
		<li>Inoculate C. albicans strain SC5314 from a frozen glycerol stock in 25 ml of YPD supplemented with Uri (2% Bacto-peptone, 1% yeast extract, 2% glucose and 80 &#181;g/ml Uridine) overnight at 30 &#176;C. 
		NOTE: Avoid growing the Candida culture more than an OD600 of 12.</li>
		<li>Take 1 ml of the C. albicans and spin it down at 2,000 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and re-suspend the pellet with 1 ml of PBS.</li>
		<li>Collect the pellet by centrifugation at 2,000 x g for 5 min at room temperature.</li>
		<li>Repeat step 2.1.4.</li>
		<li>Calculate the multiplicity of infection (MOI) of C. albicans in relation to the number of phagocytes and proceed with the phagocytosis assay as described in section 3. An OD600 of 1 is equivalent to 2.5 x 107 Candida albicans per 1 ml. 
		NOTE: Generation of Candida-BFP is described in reference13.</li>
	</ol>
	</li>
	<li>Preparation of Zymosan and IgG-coated Beads 
	NOTE: Zymosan is a &#946;-glucan-containing fungal carbohydrate particulate preparation that derived from Saccharomyces cerevisiae. Zymosan is known to evoke inflammatory signals in macrophages and dendritic cells, and it has been used extensively in phagocytosis studies 13,14,15.
	<ol>
		<li>Briefly vortex the tube that contains the particulate, and aliquot an amount sufficient for an experiment into a 1.5 ml tube. 
		NOTE: Typically we use 1:10 ratio (for one cell, add 10 zymosan particles or IgG-coated beads).</li>
		<li>Add 1 ml of ice cold PBS, and collect the particles by centrifuging for 1 min at 10,000 x g.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 1 ml ice-cold PBS.</li>
		<li>Repeat steps 2.2.2-2.2.3 
		NOTE: Proceed to step 2.2.5 or store the tube at 4 &#176;C until use.</li>
		<li>Sonicate the particulates for 10 sec in ultrasonic bath sonicator (120 V, 50-60 kHz) in order to avoid any particle aggregation.</li>
	</ol>
	</li>
</ol>
3. Phagocytosis
NOTE: Phagocytosis is a complex process that begins with binding of the particles on the cell surface of the phagocytes through interaction of PRRs with ligands on the surface of the particle. Binding is followed by the assembly of actin and its associated proteins at the site of contact, and the formation of a phagocytic cup. The subsequent actin disassembly occurs at the phagosome and results in the complete engulfment of the particulate. Below we describe the different stages of phagocytosis.
<ol>
	<li>Binding Assay 
	NOTE: The purpose of this experiment is to monitor the first step of phagocytosis that involves the interaction between the ligands on the target particles and the receptors on the phagocytes.
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide so that they are 60-70% confluent at the time of the experiment. Alternatively, plate cells on cover slips (in 24- or 12-well format) or glass-bottom 96-well plates. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Place the chamber slide (or plate) at 10 &#176;C for 10 min.</li>
		<li>Gently remove the medium from each well using a pipette or aspirator. 
		CAUTION: Perform this step carefully as each well of a chambered coverglass system is small; it is easy to perturb cells with the tip of a pipette or a strong airflow of an aspirator.</li>
		<li>Mix the fluorescently labeled particulates with a cell culture medium (prepared as described above). Add Candida-BFP (at an MOI of 10), fluorescently conjugated-zymosan or latex beads-rabbit IgG-FITC (10 particles per cell) to the cells. Use a final volume of 200 &#181;l media for one chamber well to ensure all cells are covered.</li>
		<li>Spin down the chamber slide at 277 x g for 3 min at 10 &#176;C. 
		NOTE: This centrifugation step helps to increase the contact between the particulates and the cells, and it synchronizes the binding process.</li>
		<li>Immediately transfer the chamber slide to a 37 &#176;C humidified incubator with 5% CO2 for 5 min.</li>
		<li>Remove the chamber slide from the incubator and aspirate the media.</li>
		<li>Wash the cells 3x with ice-cold PBS. 
		NOTE: Check the presence of unbound particles with a light microscope and wash more times with PBS if necessary. It is essential to remove unbound particles from the well to minimize background noise during the analysis.</li>
		<li>Fix cells by adding 500 &#181;l 4% paraformaldehyde (PFA) diluted in PBS by incubating for 30 min at room temperature. 
		CAUTION: This step requires the use of 4% PFA which is highly toxic, corrosive, and potential carcinogen. Proper caution should be taken when using this liquid. Also, protect samples from light.</li>
		<li>Wash the cells by adding 500 &#181;l of PBS into the chamber and aspirate the solution gently.</li>
		<li>Repeat 3.1.9 step twice. 
		NOTE: At this step, refill each vial immediately after removing the previous PBS.</li>
		<li>Stop the fixation by incubating with 500 &#181;l of 50 mM NH4Cl in PBS for 10 min.</li>
		<li>Wash cells twice with PBS as described in step 3.1.10.</li>
		<li>Permeabilize cells by adding 250 &#181;l binding buffer (0.1% saponin, 0.2% BSA in PBS) for 30 min at room temperature.</li>
		<li>Stain F-actin by adding fluorescently conjugated phalloidin (diluted 1:500 in binding buffer). Incubate cells for 1 hr at room temperature.</li>
		<li>Wash cells three times with PBS as described in step 3.1.10.</li>
		<li>Capture images using a confocal microscope (63X objective, and 402 nm and 488 nm laser) and analyze images using ImageJ16. 
		NOTE: A successful particle binding is characterized by the presence of a phagocytic cup at the site where the particulate contact the phagocytic cell (Figure 1). See step 3.2.5.</li>
	</ol>
	</li>
	<li>Phagocytosis Uptake Assay 
	NOTE: This experiment is designed to monitor the complete internalization of fluorescently labeled particulates by phagocytes using confocal microscopy (Figure 2).
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide so that they are 60-70% confluent at the time of the experiment. Alternatively, plate cells on cover slips (in 24- or 12-well format) or glass-bottom 96-well plates. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Keep the chamber slides (or plate) at 10 &#176;C for 10 min. 
		NOTE: Cooling is necessary in order to block endocytosis as well as unwanted phagocytosis, and subsequently to have a synchronized phagocytosis.</li>
		<li>Perform steps from 3.1.3 to 3.1.5</li>
		<li>Transfer the chamber slide to a 37 &#176;C humidified incubator with 5% CO2, and incubate for different time points. Fix cells after 30, 60 and 90 min post infection. Observe C. albicans begin to form hyphae at 60 min post infection; phagocytes begin to show cell death (pyroptosis) after 90 min8. 
		NOTE: This is a recommendation, and optimization is essential depending on the cell type and the particulate used.</li>
		<li>Perform steps from 3.1.7 to 3.1.15. Analyze images using ImageJ16. 
		NOTE: a successful uptake is characterized by a complete internalization of a particulate inside the cytoplasm of a phagocyte (Figure 2). Staining the phagocytes with cell surface marker such as phalloidin helps to delineate the contour of cells. It is also essential to perform z-stacks in order to determine whether the particulate is bound to cell surface or completely internalized.</li>
	</ol>
	</li>
	<li>Live Imaging of Phagocytosis 
	NOTE: In this protocol we describe the use of confocal microscopy to capture the different stages of phagocytosis using fluorescently conjugated zymosan. We use the dendritic cell line DC2.4 that stably expresses mCherry-tagged F-actin8, a biosensor that shows the distribution of filamentous actin in living cells. Since phagocytosis is an actin-driven process the live imaging in this cell not only enables us to capture the movement and dynamics of F-actin network, but also to visualize the different phagocytic events within the living cells (Figure 3, Movie 1).
	<ol>
		<li>Seed about 5 x 104 cells in a chamber slide at a concentration so that they are 60-70% confluent at the time of the experiment. Incubate cells overnight at 37 &#176;C humidified incubator with 5% CO2. 
		NOTE: Cells can be counted using hemocytometer or a similar technique.</li>
		<li>Keep the chamber slides on ice for 10 min. 
		NOTE: Cooling is necessary in order to block unwanted uptake, and to have a synchronized phagocytosis.</li>
		<li>Perform steps from 3.1.3 to 3.1.5</li>
		<li>Pre-warm the microscope stage to 37 &#176;C and equilibrate the chamber with 5% CO2. Wait 30-45 min for the temperature and the CO2 to stabilize before starting the imaging. 
		NOTE: Since the CO2 and temperature levels are critical for phagocytosis, it is important to turn on the microscope heater prior to the experiment to equilibrate the environmental chamber.</li>
		<li>Place the chamber slide containing the cells in the microscope incubator enclosure equilibrated at 37 &#176;C and 5% CO2.</li>
		<li>Perform live imaging17,18. 
		NOTE: The settings for the laser power (gain, pinhole, etc.) can vary depending on the type of the microscope and fluorescent probe used. Therefore, we recommend consulting the manual of the microscope for optimal condition for your live imaging 17,18.
		<ol>
			<li>Use confocal laser scanning microscope (and the associated software) for live imaging. Use 63X 1.4NA oil objective with a pinhole of 1-1.1 Airy units.</li>
			<li>To start the imaging, used the bright field to find a representative region on the chamber slide. Next, select the field by clicking the Position option on the software. To detect GFP in Track 1 used the filter set for 488 nm laser with a beam splitter at 582 nm, to detect wavelengths between 488 and 582 nm.</li>
			<li>In Track 2 select a filter set optimal for mCherry (RFP) using a beam splitter at 578 nm and detecting wavelengths between 578 and 600 nm. Use 488 nm and 578 nm lasers with 50% laser power and a gain output of 600-700. Obtain images every 30 sec for a total of 90 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+phagocytosis+in+macrophages.">Mechanisms of phagocytosis in macrophages.</a> Annu Rev. Immunol. 17, 593-623 (1999).</li><li>Flannagan, R. S., Harrison, R. E., Yip, C. M., Jaqaman, K., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+macrophage+&amp;#34;probing&amp;#34;+is+required+for+the+efficient+capture+of+phagocytic+targets.">Dynamic macrophage &amp;#34;probing&amp;#34; is required for the efficient capture of phagocytic targets.</a> J. Cell Biol. 191, 1205-1218 (2010).</li><li>Swanson, J. 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The authors declare no conflicts of interest.

Professional phagocytes, such as macrophages and dendritic cells, engulf and eliminate invading pathogens therefore making phagocytosis an important component of the host defense system. During this process phagocytes undergo extensive membrane reorganization and cytoskeleton rearrangement at their cell surface8,19,20. To better understand this dynamic process, visualization of the different stages of phagocytosis is essential. ...

Despite the continuous exposure to pathogens such as bacteria, viruses and fungi, our body is well equipped with immune mechanism that provide protection against infection. The innate immune system is the first line of defense against invading pathogens and relies mainly on phagocytic cells that recognize and internalize foreign targets.
Phagocytosis is an evolutionarily conserved cellular process that encompasses the engulfment of unwanted particulates greater than 0.5 &#181;m. Phagocytic cells express a wide range of immune receptors (also known as pattern recognition receptors, PRRs) at the cell surface that enable them to recognize pathogen-associated molecular patterns (PAMPs) present on pathogens prior to engulfment3. Pathogen binding is followed by receptor clustering at the cell surface and triggers the formation of a phagocytic cup. This results in actin-driven membrane remodeling that protrudes around the target, eventually enveloping it and pinching-off to form a discrete phagosomal vacuole2,5. The phagosome then matures and acidifies by subsequent fusion with late endosomes and lysosomes that forms phagolysosome6.
Although phagocytosis is described as receptor-mediated and actin-driven event, this process also relies on spatial-temporal modification of lipids that compose the plasma membrane, such as phosphoinositides (PIs) and sphingolipids7,8. While actin polymerization is dictated by a local accumulation of phosphoinositol-4,5-biphosphate (PI(4,5)P2) at the base of the phagocytic cup, actin depolymerization depends on the conversion of (PI(4,5)P2 to phosphoinositol-3,4,5-biphosphate (PI(3,4,5)P3)3,9. Both modifications are essential as the former leads to successful extension of pseudopods around the target and the latter enables sinking of particles in the cytosol of the phagocyte10.
Cells that have the ability to phagocytose are either professional phagocytes, like macrophages/monocytes, granulocytes/neutrophils, and dendritic cells (DCs) or nonprofessional phagocytes, such as fibroblast and epithelial cells11. Phagocytosis performed by all phagocytes plays a central role in tissue maintenance and remodeling, while phagocytosis performed by professional phagocytes is responsible for the coordination of the innate and adaptive immune response against pathogens. Professional phagocytes do not only engulf and kill the pathogen, but also present antigens to the lymphoid cells of the adaptive immune system. This contributes to the release of pro-inflammatory cytokines and to the engagement of lymphoid cells, therefore leading to the successful blockade of infection12.
Conventional biochemical techniques have been instrumental in gaining knowledge regarding the molecular mechanism of different cellular processes during phagocytosis, such as post-translational modifications and various high-affinity associations between proteins. However, it is difficult to obtain information regarding the spatial and temporal dynamics of phagocytic events using the conventional biochemical methods. Live cell imaging not only allows us to monitor cellular events in a time sensitive manner but also enables us to gain information at a single cell level. Here we describe a method to investigate the different stages of phagocytosis, as well as to analyze the whole process spatiotemporally using confocal microscopy.

<table><tbody><tr><td>&amp;beta;-Mercaptoethanol</td><td>AppliChem</td><td>A1108</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9998</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dilactate)</td><td>ThermoFisher</td><td>D3571</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>ThermoFisher</td><td>BP-231-1</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Minimal Eagle&amp;rsquo;s medium)</td><td>Gibco</td><td>11965</td><td></td></tr><tr><td>PBS, 1x (Phosphate- Buffered Saline)</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td></td></tr><tr><td>FITC-coupled IgG-coated latex beads</td><td>Cayman</td><td>500290</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>Paraformaldehyde Solution (4% in PBS)</td><td>Affymetrix</td><td>19943 1 LT</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/gibco-penicillin-streptomycin-10-000-u-ml-3/15140122&quot;&gt;15-140-122&lt;/a&gt;</td><td></td></tr><tr><td>Phalloidin-Alexa Fluor 488</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>RAW 264.7 cells</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>RPMI (Roswell Park Memorial Institute)</td><td>Gibco</td><td>61870</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>S7900</td><td></td></tr><tr><td>Trypan blue solution (0.4% (w/v) in PBS)</td><td>Corning cellgro</td><td>MT25900CI</td><td></td></tr><tr><td>Trypsin-EDTA (1x) (0.05%)</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Tween 20 Surfact-Amps Detergent Solution</td><td>ThermoFisher</td><td>&amp;nbsp;85114</td><td></td></tr><tr><td>Zymosan-Alexa Fluor 594</td><td>ThermoFisher</td><td>Z23374</td><td></td></tr><tr><td>Chambered 1.0 Borosilicate Coverglass system (8 chambers)</td><td>ThermoFisher</td><td>155361</td><td></td></tr><tr><td>Glasstic slide 10 with grids</td><td>Hycor</td><td>87144</td><td></td></tr></tbody></table>

visualizing the early stages of phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Microscope-based method to monitor the different stages of phagocytosis is presented. The different events during the phagocytosis of various fluorescent particulates by DC2.4 cells are shown. Using the techniques described here, we investigated the role of sphingolipids in the early stages of phagocytosis. For this purpose, DC2.4 dendritic cells genetically deficient in Sptlc2, the enzyme that catalyzes the first and rate-limiting step in the sphingolipid biosynthetic pathway, were used. As compared to wild type cells, ...

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Visualizing the Early Stages of Phagocytosis

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

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Biology

16.6K Views.  Oregon Health & Science University. Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

Video: Visualizing the Early Stages of Phagocytosis

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Immunology and Infection

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

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 ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

Visualizing the Early Stages of Phagocytosis

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