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EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Note: The plasmid used Lifeact-mCherry is a kind gift of Dr. Guillaume Montagnac, Institut Curie, Paris.
1. Cells and Transfection
Note: RAW264.7 macrophages are grown to subconfluency 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 electroporation. Routinely approximately 5 - 6 x 106 cells are transfected with 20 &#181;g or 10 &#181;g of plasmid for each transfection or co-transfection respectively. Note that other means of transfection based on electroporation or lipofection can be used as alternative approaches.
<ol>
	<li>Scrape the cells with a cell lifter and resuspend them in 10 ml of culture medium by pipetting up and down several times.</li>
	<li>Centrifuge the cell suspension 300 x g, 5 min at RT in swinging angle rotor.</li>
	<li>Preheat 10 ml of complete medium supplemented with 10 &#181;g/ml of gentamicin at 37 &#176;C.</li>
	<li>Following centrifugation, discard the supernatant and resuspend the pellet in 3 ml of &#34;washing buffer A&#34; from the electroporation kit.</li>
	<li>Centrifuge the cell suspension 300 x g, 5 min at room temperature in swinging angle rotor.</li>
	<li>In the meantime prepare the DNA mix at room temperature: 120 &#181;l 2x Buffer B, 20 &#181;g of plasmid DNA coding for the protein of interest, q.s.p. 240 &#181;l H2O.</li>
	<li>Following centrifugation, discard the entire supernatant.</li>
	<li>Resuspend the cell pellet in the DNA mix and transfer into 4 mm electroporation cuvettes.</li>
	<li>Incubate at RT for 3 min.</li>
	<li>Electroporate at 250 V, 900 &#181;F.</li>
	<li>Immediately resuspend the cells in the pre-warmed (37 &#176;C) complete medium supplemented with gentamicin and plate them in a 100 mm dish.</li>
	<li>Incubate the transfected cells overnight at 37 &#176;C, 5% CO2.</li>
	<li>The next morning, replace the complete medium with 10 ml of serum-free microscopy medium (RPMI 1640 without phenol red, 10 mM HEPES, 1 mM sodium pyruvate, 50 &#181;M &#946;-mercaptoethanol, 2 mM L-Glutamine).</li>
</ol>
2. Opsonization of Red Blood Cells
Note: As a model of particle target for macrophages, sheep red blood cells (SRBCs) are used. Usually, around 7 x 106 SRBCs per 35 mm glass bottom dish is used.
<ol>
	<li>Wash the SRBCs with 100 &#181;l of 1x PBS (phosphate buffered saline)/0.1% BSA (bovine serum albumin) and centrifuge (600 x g, 4 min).</li>
	<li>Following centrifugation, discard the supernatant and resuspend the SRBCs in 100 &#181;l of 1x PBS/0.1% BSA and centrifuge (600 x g, 4 min).</li>
	<li>Following centrifugation, discard the supernatant and resuspend the SRBCs in 1x PBS/0.1% BSA with rabbit IgG anti-SRBCs at sub-agglutinating concentration. Use 500 &#181;l of solution containing antibody/5 &#181;l of SRBCs. 
	Note: The sub-agglutinating concentration of antibodies corresponds to the lowest concentration that did not induce agglutination detected as formation of a network. In general, the sub-agglutinating concentration is 8.2 &#181;g/ml.
	<ol>
		<li>Determine the concentration of IgG anti-SRBCs required to opsonize the SRBCs by a hemagglutination test. Serially dilute the IgG anti-SRBCs (stock at 13.1 mg/ml) between 1/50 - 1/25,600 in a microplate. Add 2 x 106 SRBCs in each well. Incubate the plate in the dark at RT for several hr.</li>
	</ol>
	</li>
	<li>Incubate at RT for 30 mins with slow rotation.</li>
	<li>After two washes in 1x PBS/0.1% BSA as described above, resuspend the IgG-opsonized SRBCs (IgG-SRBCs) in pre-warmed serum-free microscopy medium (2 ml/dish).</li>
</ol>
3. Poly-lysine Coating of Coverslips
<ol>
	<li>In the meantime, treat 35 mm glass bottom dishes with 2 ml of 0.01% poly-L-lysine for 30 min at room temperature.</li>
	<li>Wash the dishes two times with 2 ml of 1x PBS.</li>
</ol>
4. Non-covalent Fixation of SRBCs on Glass Bottom Dishes
<ol>
	<li>Pour 2 ml of SRBCs suspension per 35 mm glass bottom dish.</li>
	<li>Centrifuge with a swinging rotor at 500 x g during 2 min onto 35 mm glass bottom dishes.</li>
	<li>Remove the supernatant and wash once with 2 ml of 1x PBS/10% BSA.</li>
	<li>Incubate the particles for 30 min with 2 ml of 1x PBS/10% BSA per dish.</li>
	<li>Wash the dishes three times with 2 ml of 1x PBS.</li>
	<li>Replace 1x PBS with 2 ml of pre-warmed (37 &#176;C) serum-free microscopy medium.</li>
</ol>
5. Phagocytosis Visualized by TIRFM
<ol>
	<li>Microscope 
	Note: TIRFM was performed using a microscope equipped with an oil-immersion objective (N 100X, NA1.49), a heating chamber with CO2, and two single photon detection cameras EMCCD (Electron Multiplying Charge Coupled Device) coupled with a 1.5X lens.
	<ol>
		<li>The day before the TIRF microscope session, turn on the heating chamber at 37 &#186;C to allow a homogeneous heating of the microscope stage.</li>
	</ol>
	</li>
	<li>Critical Angle Determination 
	Note: ImageJ Color Profiler software is used to process TIRF image streams.
	<ol>
		<li>Place a 35 mm glass bottom dish containing opsonized SRBC with serum-free microscopy medium under the microscope.</li>
		<li>Scrape the cells and resuspend them in the medium before adding them (100 - 500 &#181;l) in the dish.</li>
		<li>Use the &#34;Live Acquisition&#34; software to control the microscope and perform excitation with a 491 nm and/or a 561 nm laser to identify cells expressing fluorescently tagged proteins.</li>
		<li>Identify a cell that expresses the fluorescently tagged proteins.</li>
		<li>Place it in the middle of the field and acquire 500 images at one excitation wavelength, with different angles starting from 0&#186; up to 5&#186;, with an increment of 0.01&#186; (Figure 1A). The angles are automatically changed by the microscope system.</li>
		<li>Determine the critical angle allowing the incident light to be totally reflected at the glass/ medium interface and generate the evanescent wave. Using ImageJ Color Profiler software, open the image sequence by clicking on &#34;File&#34;, &#34;Open&#34; and select the file.</li>
		<li>Select a region of interest (ROI), with the rectangular tool, in the cell with a uniform fluorescence (Figure 1B yellow 1).</li>
		<li>Plot the &#34;Z axis profile&#34; mean fluorescence intensity measured in the ROI with function of the angles on the x axis by clicking on &#34;Image&#34; tab, &#34;Stacks&#34; in the drop down menu and &#34;Plot Z-axis Profile&#34; (Figure 1B red 2). 
		Note: The critical angle is the angle leading to the maximum fluorescence before a sharp decrease in fluorescence.</li>
		<li>Use any value of angle on the x-axis superior to the critical angle during the microscopy session to obtain a TIRF signal as example 2.00 (Figure 1C).</li>
	</ol>
	</li>
	<li>Acquisitions
	<ol>
		<li>Using Live Acquisition software, set up the parameters of acquisition with the module &#34;Protocol Editor&#34; (Figure 2A).
		<ol>
			<li>Create a protocol with a &#34;loop&#34; comprising &#34;Multi Channels&#34; acquisition to acquire fluorescent signal from proteins of interest in the TIRF region. Enter the TIRF angle (for example 2.00), the exposure time (for example 50 msec) and the laser intensity (for example 50%) (Figure 2B 1).</li>
			<li>Introduce a &#34;Z move&#34; of the objective 3 &#181;m above the TIRF region to obtain signal acquisition in epifluorescence with the &#34;Multi Channels &#34;tool. Enter an angle below the critical angle (as example 1.00), the time of exposition (50 msec) and the laser intensity (50%) (Figure 2B, 2 and 3).</li>
			<li>Next add a &#34;z move&#34; of the objective 3 &#181;m below, to return in the TIRF region (Figure 3B 4). Add a &#34;snapshot&#34; of the cell in bright light LED (Light-Emitting Diode) (Figure 2B 5).</li>
		</ol>
		</li>
		<li>Find a cell of interest with a moderate level of fluorescently tagged protein expression that initiates phagocytosis of SRBC by extending plasma membrane around the particle.</li>
		<li>Place it in the middle of the field.</li>
		<li>Start streaming acquisition of 500 - 1,000 frames. In the &#34;loop count&#34; tab, enter &#34;750 frames&#34; (Figure 2B 1). 
		Note: ImageJ Color Profiler software is used to process TIRF image streams.</li>
		<li>Open the sequence images in Image J software by clicking &#34;File&#34;, &#34;Open&#34; and choosing the file.</li>
		<li>Separate the channels by clicking on the tab &#34;Image &#34;, &#34;Hyperstacks&#34; drop down menu and on &#34;Stack to hyperstack&#34; function. Complete the appeared window: Order: xyctz; Channel (c): number of channels (ex: &#34;2&#34; if you have two fluorochromes); Slices (z): number of slices in z axis (ex: &#34;1&#34; if there is no movement in the z- axis); Frames (t): number of images divided per number of channels; Display mode: Grayscale. 
		Note: Two separate image sequences are generated, corresponding to the two different channels.</li>
	</ol>
	</li>
</ol>

<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%</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</td>
			<td>TILL Photonics</td>
			<td></td>
			<td>Oil-immersion objective (N 100x, NA1.49.), 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</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium GLUTAMAX 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>

tirf microscopy-based visualization of phagosome formation and closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

<img alt="Figure 1" src="/files/ftp_upload/22014/22014_Figure1.jpg" /> 
Figure 1. Schematic Representation of the &#34;Phagosome Closure Assay&#34; Analyzed by TIRFM. The phagosome closure assay is performed using macrophages transiently expressing one or two fluorescently tagged protein. Macrophages are deposited on IgG-opsonized SRBCs non-covalently fixed on poly-lysine coated coverslips. Images are recorded in TIRF mode to detect the site of phagosome closure and ...

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Note: The plasmid used ...

Take a polymer-coated glass bottom dish containing IgG-opsonized red blood cells or RBCs adhered to its surface.
Place the dish on a total internal reflection fluorescence microscope stage, maintaining it at physiological temperature.
Add macrophages containing fluorescently labeled cytoplasmic peptides that bind to polymerized actin, facilitating cytoskeleton visualization.
During imaging, direct a laser beam at an angle superior to the critical angle, causing internal reflection and evanescent wave generation at the contact point.
The evanescent waves selectively illuminate the fluorophores at the glass-sample interface, allowing real-time visualization of phagocytosis.
Macrophage Fc receptors bind to the IgG-opsonized RBC, causing receptor clustering around the contact area.
Clustering triggers intracellular signaling pathways and GTPase activation, driving actin polymerization and dynamin recruitment, forming pseudopods.
The pseudopods extend around the RBC, forming a phagocytic cup.
Eventually, the pseudopod tips fuse. The accumulated dynamin mediates phagosome separation from the cell membrane, internalizing the opsonized RBC.

tirf-microscopy-based-visualization-of-phagosome-formation-and-closure

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

76 vues. Source : Marie-Anaïs, F., et al. ""Essai de fermeture du phagosome » pour visualiser la formation des phagosomes en trois dimensions à l’aide de la microscopie fluorescente à réflexion interne totale (TIRFM). J. Vis. Exp. (2016). Cette vidéo présente une technique de microscopie à fluorescence par réflexion interne totale (TIRF) à haute résolution pour la visualisation en temps réel de la formation et de la fermeture des phagosomes pendant la phagocytose médiée par les macropha...

Vidéo: Visualisation de la formation et de la fermeture des phagosomes par microscopie TIRF - Concept

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.

Time-lapse imagerie 3D de phagocytose par les Macrophages de souris

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

Recherche

JoVE Journal

Immunologie et infection

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.

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.

Visualiser les premiers stades de la phagocytose

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

Biologie

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

Évaluation de la capacité phagocytique spécifique à l’âge des hémocytes adultes de Drosophila melanogaster à l’aide d’un test de phagocytose in vivo

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Transcription

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure