We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nad&#232;ge Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales" (ANRS) including a doctoral fellowship for JM.

Note: The plasmid used Lifeact-mCherry is a kind gift of Dr. Guillaume Montagnac, Institut Curie, Paris, generated after 17.
1. Cells and Transfection
Note: RAW264.7 macrophages are grown to sub confluency in complete medium (RPMI (Roswell Park Memorial Institute) 1640 medium, 10 mM HEPES, 1 mM sodium pyruvate, 50 &#181;M &#946;-mercaptoethanol, 2 mM L-Glutamine and 10% FCS (Fetal Calf Serum)) in a 100 mm plate. They are transfected with plasmids encoding fluorescently tagged proteins by electropor...

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+during+phagocytosis.">Information processing during phagocytosis.</a> Nat Rev Immunol. 12, 492-502 (2012).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu Rev Immunol. 20, 825-852 (2002).</li><li>Canton, J., Neculai, D., 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=Scavenger+receptors+in+homeostasis+and+immunity.">Scavenger receptors in homeostasis and immunity.</a> Nat Rev Immunol. 13, 621-634 (2013).</li><li>Freeman, S. A., 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=Phagocytosis:+receptors,+signal+integration,+and+the+cytoskeleton.">Phagocytosis: receptors, signal integration, and the cytoskeleton.</a> Immunol Rev. 262, 193-215 (2014).</li><li>Scott, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4,5-bisphosphate+hydrolysis+directs+actin+remodeling+during+phagocytosis.">Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.</a> J Cell Biol. 169, 139-149 (2005).</li><li>Schlam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase+enables+phagocytosis+of+large+particles+by+terminating+actin+assembly+through+Rac/Cdc42+GTPase-activating+proteins.">Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.</a> Nat Commun. 6, 8623 (2015).</li><li>Marion, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF-kappaB+Signaling+Protein+Bcl10+Regulates+Actin+Dynamics+by+Controlling+AP1+and+OCRL-Bearing+Vesicles.">The NF-kappaB Signaling Protein Bcl10 Regulates Actin Dynamics by Controlling AP1 and OCRL-Bearing Vesicles.</a> Dev Cell. 23, 954-967 (2012).</li><li>Loovers, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+phagocytosis+in+Dictyostelium+by+the+inositol+5-phosphatase+OCRL+homolog+Dd5P4.">Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4.</a> Traffic. 8, 618-628 (2007).</li><li>Deschamps, C., Echard, A., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+cytokinesis:+do+cells+use+common+tools+to+cut+and+to+eat?+Highlights+on+common+themes+and+differences.">Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences.</a> Traffic. 14, 355-364 (2013).</li><li>Johnson, D. S., Jaiswal, J. K., Simon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12,+Unit+12.29:+Total+internal+reflection+fluorescence+(TIRF)+microscopy+illuminator+for+improved+imaging+of+cell+surface+events.">Chapter 12, Unit 12.29: Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.</a> Curr Protoc Cytom. , (2012).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Marie-Anais, F., Mazzolini, J., Herit, F., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-actin+cross-talk+contributes+to+phagosome+formation+and+closure.">Dynamin-actin cross-talk contributes to phagosome formation and closure.</a> Traffic. 17 (5), 487-499 (2016).</li></ol>

The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 &#176;C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We notice...

Phagocytosis is a major cell function that starts with the recognition and binding of material to surface receptors, which then leads to the internalization and degradation of the ingested material. While single-celled eukaryotes such as the mold Dictyostelium discoideum and amoebae use phagocytosis for feeding on bacteria, higher organisms have evolved with professional cells. Macrophages or dendritic cells are the first line of defense against pathogens in various tissues and organs, and are crucial to activate the adaptive immune system through antigen presentation and cytokine production 1-4. Under certain circumstances phagocytosis can be performed by non-professional phagocytic cells, e.g., endothelial and epithelial cells. This process is important to maintain homeostasis during development and in adulthood for normal tissue turnover and remodeling. Finally, specialized phagocytes such as Sertoli cells in the testis or retinal pigment epithelial cells are extremely potent phagocytes 5.
The formation of a phagosome where degradation of microorganisms or cellular debris occurs starts with the clustering of phagocytic receptors on the surface of the phagocytic cell. Downstream signaling events following clustering of opsonic receptors such as Fc receptors (FcR) or complement receptors (CRs) have been well characterized. However, there are also numerous non-opsonic receptors including Toll-like receptors (TLRs), lectins, mannose receptors and scavenger receptors. These receptors recognize determinants on the particle surface such as mannose or fucose residues, phosphatidylserine, and lipopolysaccharides 1,6-9.
Pathogen or cell debris recognition involves binding to and clustering of several types of phagocytic receptor, which then lead to intense and transient actin remodeling. In parallel, focal exocytosis of intracellular compartments contributes to the release of membrane tension and is important for efficient phagocytosis of large particles. The signaling events leading to actin polymerization and membrane deformation were dissected in experimental models that triggered a single phagocytic receptor. During FcR-mediated phagocytosis, there is intense actin polymerization that is regulated by small GTPases (Rac, Cdc42). Among their downstream effectors, the Wiskott-Aldrich syndrome protein (WASP) leads to activation of the Actin-Related Protein 2/3 complex (Arp2/3) that nucleates actin filaments 1,2,4,10. Local production of phosphatidylinositol-4, 5-bisphosphate (PI(4,5)P2) is crucial for initial actin polymerization that drives pseudopod formation. Its conversion to PI(3,4,5)P3 is required for pseudopod extension and phagosome closure 11. Several pathways contribute to the disappearance of PI(4,5)P2. Firstly detachment of phosphatidylinositol phosphate kinases (PIPKIs) from the phagosome arrests PI(4,5)P2 synthesis. Secondly, it can be phosphorylated and consumed by class I PI3K kinases (PI3K) and converted in PI(3,4,5)P3 12. A role for phosphatases and phospholipases has also been implied in PI(4,5)P2 hydrolysis and F-actin removal during phagocytosis in mammalian cells and in Dictyostelium13,14. The phospholipase C (PLC)&#948; hydrolyzes PI(4,5)P2 into diacylglycerol and inositol-1,4,5-trisphosphate. The PI(4,5)P2 and PI(3,4,5)P3 phosphatase OCRL (oculocerebrorenal syndrome of Lowe) has also been implicated in phagosome formation. Precise local formation of F-actin and its depolymerization is tightly regulated in space and time and we have shown that recruitment of intracellular compartments is important to deliver locally the OCRL phosphatase, thus contributing to local actin depolymerization at the base of the phagocytic cup 13,15. For this, we used the experimental set up described here.
The mechanism and molecular players required for phagosome closure and membrane scission remain poorly defined because of the difficulties in visualizing and monitoring the site of phagosome closure. Until recently, phagocytosis was observed on fixed or living cells that internalize particles on their dorsal face or on their sides, making the timely visualization of the site of phagosome closure difficult. In addition, fixing methods could cause retraction of membranes and bias the results on pseudopodia extension and closure. By contrast, the assay that we have set up and describe here allows us to visualize pseudopod extension and the closure step of phagocytosis in living cells 13, based on total internal reflection microscopy (TIRFM) 16. This optical technique uses an evanescent wave to excite fluorophores in a thin area at the interface between a transparent solid (coverslip) and a liquid (cell culture medium). The thickness of the excitation depth is around 100 nm from the solid surface, allowing visualization of molecular events close to the plasma membrane. TIRFM allows a high signal-to-background ratio, and limits the out-of-focus fluorescence collected and the cytotoxicity due to illumination of cells.
Taking advantage of TIRFM, we developed the &#34;phagosome closure assay&#34; in which coverslips are activated with poly-lysine and then coated with IgG-opsonized red blood cells (IgG-SRBCs). Macrophages expressing transiently fluorescently tagged proteins of interest are then allowed to engulf the IgG-SRBCs. While cells detach the target particles that are non-covalently bound to the glass surface, the tips of the pseudopods can be observed and recorded in the TIRF mode. TIRF acquisitions are combined with acquisitions in the epifluorescence mode, after shifting the stage 3 &#181;m above, which allows the visualization of the base of the phagocytic cup. Addition of pharmacological drugs such as those inhibiting actin or dynamin during the process is also possible to further dissect the process at the molecular level.
The protocol described in detail here is for a RAW264.7 murine macrophage cell line and opsonized particles, but virtually, it can be adapted to any other phagocytic cell and with other targets such as beads. This method will allow better characterization of the regulation in time and space of the molecular players involved in pseudopod extension and phagosome closure during various phagocytic processes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-sheep red blood cells IgG</td>
			<td>MP Biomedicals</td>
			<td>55806</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes 4mm</td>
			<td>Cell project</td>
			<td>EP104</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fischer Scientific</td>
			<td>14190-094</td>
			<td>Room temperature</td>
		</tr>
		<tr>
			<td>Electrobuffer kit</td>
			<td>Cell project</td>
			<td>EB110</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-Treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene X-cell pulser</td>
			<td>Biorad</td>
			<td>165-2661</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C Case</td>
			<td></td>
		</tr>
		<tr>
			<td>iMIC&#160;</td>
			<td>TILL Photonics</td>
			<td></td>
			<td>&#160;Oil-immersion objective&#160;(N 100x, NA1.49.),&#160; heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine Solution 0.1%</td>
			<td>Sigma</td>
			<td>P8920-100ml</td>
			<td>Dilution at 0.01% in water&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td>Life technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td>Life technologies</td>
			<td>11835-105</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Sheep red blood cells (SRBCs)</td>
			<td>Eurobio</td>
			<td>DSGMTN00-0Q</td>
			<td>Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
	</tbody>
</table>

"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.

The experimental system described in this manuscript is schematically represented in Figure 1. Transfected RAW264.7 macrophages expressing the proteins of interest fused to a fluorescent tag are placed into contact with IgG-opsonized sheep red blood cells (SRBCs) that were non-covalently fixed on the coverslip. The macrophages can detach the SRBC from the coverslip to engulf it. The TIRF microscope used allows concomitant acquisition of signals from the TIRF area correspo...

Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

&quot;Phagosome Closure Assay&quot; 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.

"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 ...

The overall goal of this protocol is to visualize phagosome formation, as well as the precise site of phagosome schism in 3D, in living macrophages, using Total Internal Reflection Florescence, or TIRF Microscopy. This method combines TIRF Microscopy with epifluorescence to monitor the step by step localization of two different fluorescent proteins during pseudopod extension and tip fusion. The technique provides a systematic method to determine the critical angle and obtain a TIRF signal which is crucial for drawing conclusions about the close to the plasma membrane.The day before the TIRF microscope session, turn on the heating chamber to 37 degrees Celsius to allow a homogeneous heating of the microscope stage. The next morning, using 100 microliters of PBS, supplemented with 0.1%BSA to wash 7x10 to the 6th sheep red blood cells or SRBCs per 35-millimeter glass-bottom dish two times. After the second centrifugation, resuspend the cells in 500 microliters of rabbit IGG anti-SRBCs, at a sub-agglutinating concentration, in PBS supplemented with 0.1%BSA per 5 microliters of cells, and incubate the cells for 30 minutes with a slow rotation.At the end of the incubation, wash the SRBCs two times in PBS plus BSA, and resuspend the cells in 2 milliliters of 37-degree Celsius, serum-free microscopy medium per dish. Then, treat 35-millimeter glass-bottom dishes with 2 milliliters of 0.1%poly-L lysine for 30 minutes at room temperature, followed by two washes with 2 milliliters of PBS. For non-covalent fixation of the SRBCs, pour 2 milliliters of the IGG opsonized cells into each of the polylysine-coated dishes and centrifuge the samples in a swinging rotor centrifuge.Discard the supernatants and wash the particles one time with 2 milliliters of PBS plus 10%BSA. Then, incubate the particles with 2 milliters of fresh PBS plus 10%BSA for 30 minutes at room temperature, followed by three washes with 2 milliliters of PBS. After the last wash, replace the PBS with 2 milliliters of 37-degree Celsius, serum-free microscopy medium.Place an SRBC-coated dish onto the microscope stage. Then, scrape the transfected cells of interest from the bottom of the culture dish, and pipette the cells a few times to achieve a single-cell suspension. Add the transfected cells to the SRBC-coated dish.Start the live acquisition software. Find a cell that expresses the fluorescently-tagged proteins, and adjust the position of the dish so that a cell of interest is in the middle of the field. Acquire 500 images at different angles from zero to 5%at 0.01 degree increments, at one excitation wavelength.To determine the critical angle for the incident light to be totally reflected at the glass-medium interface, and generate an evanescent wave, open the image sequence in the appropriate imaging software. Select a region of interest in the cell with uniform fluorescence. Then, under the Image tab, select Stacks, and Plot Z Axis Profile.To plot the z axis profile mean fluorescence intensity measured in the region of the interest with the function of the angles on the x axis. Any angle value on the x axis, superior to the critical angle, can then be used during the microscopy session to obtain a TIRF signal. Next, in the live acquisition software, use the protocol editor to set the parameters of the acquisition.Including the number of loop acquisition sequences, the fluorescent channels and their laser intensity, the TIRF angle, and the exposure time. Set the Z move of the objective to reach the epifluorescence mode plane. Then, in the epifluorescence mode, set the TIRF angle to 1, and the Z move of the objective back to the TIRF plane.Then, obtain an image of the cell in the bright light LED mode. Now, find a cell of interest with a moderate level of fluorescently-tagged protein expression, engaged in phagocytosis with an SRBC, and move the cell into the middle of the field. Note that such a cell can be identified by an extended plasma membrane around the particle.Enter the number of frames in the Loop Count tab to begin a streaming acquisition of 500 to 1, 000 frames. If necessary, change the laser intensity and exposure time to adapt to the different levels of fluorescence in the cell. To generate two separate image sequences corresponding to the two channels, open the sequences in the imagine software and click on the Image tab.In the Hyperstacks menu, select Stack to Hyperstack. Then, in the pop-up window, enter xyctz for the Order, the number of fluorochromes used for the Channels, the number of Slices in the z axis, the number of images divided by the number of channels under Frames, and Grayscale for the Display Mode. Then, split the channels.Here, a representative live-cell TIRF microscopy movie of a phagosome closure assay in raw 264.7 macrophages engulfing an IGG opsonized SRBC is shown. As the tips of the pseudopods opposed to the glass coverslip around the SRBC, an F-actin ring is detected in the TIRF area that progressively narrows until it closes. In parallel, the blurry F-actin signal detected by the epifluroescence, after shifting the stage three microns above the TIRF area corresponds to de-polymerization at the base of the phagocytic cup.After three minutes, the SRBC is totally internalized, as confirmed by transmitted light imaging. Using this method, the of actin at the base of the phagocytic cup were the focal exocytosis of the intracellular compartments occurs, can be demonstrated. After its acquisition, a cell can be further processed for correlative electron microscopy, or the whole cell population can be fixed and frozen processed for classical immunofluorescence microscopy.It is important to keep in mind that phagocytosis is very sensitive to temperature, and therefore, the temperature within the heating chamber and the culture dish medium should be maintained at a constant 37 degrees Celsius temperature throughout the procedure. After watching this video, you should have a good understanding of how to opsonize particles, coat cover slips with the particles, determine the critical angles for TIRF microscopy, and image the localization of proteins of interest during live cell phagocytosis.

phagosome-closure-assay-to-visualize-phagosome-formation-three

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Immunology and Infection

6.3K vistas.  Université Paris Descartes, Sorbonne Paris Cité. The overall goal of this protocol is to visualize phagosome formation, as well as the precise site of phagosome schism in 3D, in living macrophages, using Total Internal Reflection Florescence, or TIRF Microscopy. This method combines TIRF Microscopy with epifluorescence to monitor the step by step localization of two different fluorescent proteins during pseudopod extension and tip fusion. The technique provides a systematic method to determine the critical angle and obtain a TIRF signal whi...