eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Peripheral Blood Mononuclear Cell Isolation
<ol>
	<li>Obtain human whole blood from a healthy donor or a patient via venipuncture using vacutainers containing acid-citrate-dextrose (ACD) anticoagulant (yellow-top tubes). 
	NOTE: Whole blood can be stored in ACD at room temperature (18-22 &#176;C) for up to 36 hours before proceeding to the next step. Normally, 1-2 10-mL vacutainer tubes of whole blood are sufficient for the assay.</li>
	<li>Dilute the whole blood 1:1 v/v in warm complete RPMI medium (RPMI-1640 supplemented with 10% fetal bovine serum, 20 mM HEPES, and 0.01 mg/mL gentamicin).</li>
	<li>Isolate peripheral blood mononuclear cells (PBMCs) from the diluted whole blood using density gradient centrifugation, as recommended by the manufacturer (See List of Materials). Layer the diluted blood very slowly over the density gradient (warmed to room temperature, 18-22 &#176;C). 
	NOTE: Minimize the amount of mixing at the interface for the optimal separation of blood by carefully layering the blood mixture in a dropwise fashion or by using a pipette.
	<ol>
		<li>Allow the blood mixture to slowly layer over the top of the density gradient by placing the pipet tip close to the density gradient and by enabling the blood mixture to run down the side of the tube very slowly.</li>
		<li>Depending on the scale of the experiment, layer 10 mL of blood mixture on top of 3 mL of density gradient (in a 15-mL tube) or layer 35 mL of blood mixture on top of 15 mL of density gradient (in a 50-mL tube). Typically, 10 mL of whole blood yields 10 million PBMCs, with some donor-to-donor variation. 
		NOTE: It is very important that there is no mixing of the blood mixture with the density gradient. The blood mixture should layer over the density gradient and slowly rise until all the blood is on top of the density gradient.</li>
		<li>Centrifuge the layered mixture at 700 x g for 30 min without brakes. The centrifuged mixture should be separated into 5 layers (from top to bottom): plasma, buffy coat (containing PBMCs), density gradient material, granulocytes, and RBCs.</li>
		<li>Remove and discard the majority of the plasma and carefully retrieve the buffy coat (PBMC) content into a new 15-mL tube using a Pasteur pipette and a suction bulb. 
		NOTE: The removal of the buffy coat layer is effectively done by applying suction on the Pasteur pipette while performing a circular motion around the outside of the layer, with the tip of the pipette against the tube.</li>
	</ol>
	</li>
	<li>Wash the isolated PBMCs three times in pH 7.3 phosphate-buffered saline (PBS) by centrifuging at 350 x g for 10 min (with full brakes) in between washes. Reconstitute the PBMC pellet in complete RPMI medium. Depending on the size of the pellet, 3-7 mL of medium is sufficient.</li>
	<li>Count the PBMC using trypan blue and a hemocytometer. Only count those cells that are not stained by the trypan blue. Reconstitute the PBMCs to 1,750,000 cells/mL in a complete RPMI medium.</li>
	<li>Seed 400 &#181;L (700,000 cells) into each well of an 8-chamber slide. Incubate the slide in a 37 &#176;C, fully humidified tissue culture incubator (supplemented with 5% CO2) for 1 h to allow the monocyte/macrophages to adhere.</li>
</ol>
2. Pre-treatment of Adhered Monocytes
NOTE: This step is only necessary if looking for ways to inhibit or enhance phagocytosis.
<ol>
	<li>Pre-treat adhered monocytes with any drug(s) or compound(s) of interest. Reconstitute the drug(s) or other test material to the desired concentration using a complete RPMI medium. 
	NOTE: For example, 200 &#181;g/mL of IVIG is typically used to yield a 95-100% inhibition of phagocytosis when using human monocytes.</li>
	<li>Aspirate and discard the supernatant containing any non-adherent cells from the 8-chamber slide after the 1-h incubation (after step 1.6). Replace it with 400 &#181;L of a drug or another treatment and incubate for 1 h at 37 &#176;C.
	<ol>
		<li>When aspirating and replacing solutions in the 8-chamber slide, make sure to control the flow of the fluids so that weakly adhered cells do not lift off. Also, work with only 2-3 empty wells at a time and avoid drying the wells. 
		NOTE: Typically, each treatment is performed in technical triplicates.</li>
	</ol>
	</li>
</ol>
3. Opsonization of R2R2 Red Blood Cells
NOTE: Opsonized R2R2 RBCs are used as a positive control for Fc&#947;R-mediated phagocytosis. Na&#239;ve R2R2 should be stored in Alsever&#39;s solution (to prolong shelf life) at 4 &#176;C for up to 1 month. Alsever&#39;s solution is made in-house and composed of 0.8% w/v trisodium citrate (dihydrate), 1.9% w/v dextrose, 0.42% w/v sodium chloride, and 0.05% w/v citric acid (monohydrate). If R2R2 cells are not available, other Rh-phenotyped cells, such as R1R2, R1R1, R1r, or R2r, can be used.
<ol>
	<li>Wash R2R2 (cDE/cDE) red blood cells in PBS a total of three times using centrifugation at 350 x g for 5 min each. 
	NOTE: The amount of RBCs needed depends on the experimental size and can be back-calculated. Always wash an excess amount of RBCs, since RBCs are lost during each wash due to lysis or during the removal of supernatant. For example, in an experiment with 5 treatments and 2 controls, all conducted in technical triplicates, there is a total of 21 wells. 21 wells with 400 &#181;L/well of 1.25% RBC mixture indicate that 105 &#181;L of packed, opsonized RBC is needed. 200 &#181;L of RBC should be initially washed and 150 &#181;L should be opsonized to ensure that there is an adequate amount of RBCs for the phagocytosis step.</li>
	<li>Opsonize the washed R2R2 pellet with 1:1 v/v polyclonal anti-D antibodies from human serum and incubate for 1 h at 37 &#176;C, with intermittent mixing. 
	NOTE: If polyclonal anti-D antibodies are not available, it is possible to use monoclonal anti-D antibodies, which are commercially available, or anti-D that are normally used for Rh immune prophylaxis, which can be obtained from blood banks or transfusion services. Optimization testing should be conducted at the beginning when setting up the assay, and whenever switching lots of un-titered polyclonal antibodies or switching to a monoclonal antibody. This is to identify the optimal concentration or volume of anti-D required for an antiglobulin test (IAT) result of between 3+ and 4+ and a phagocytosis result of between 70-90 phagocytosed RBCs per 100 monocytes counted.</li>
	<li>Wash the opsonized R2R2 a total of three times in PBS using centrifugation at 350 x g for 5 min each. 
	NOTE: Successful opsonization of R2R2 can be confirmed by performing an indirect IAT. Briefly, secondary polyclonal anti-human antibodies are added to bind primary opsonizing antibodies on RBC surfaces, and the amplified signal can be observed in the form of hemagglutination. A detailed manufacturer protocol can be found in the supplementary materials file.</li>
	<li>Reconstitute the washed R2R2 pellet to 1.25% v/v using a complete RPMI medium. 
	NOTE: Excess opsonized R2R2 can be stored in Alsever&#39;s solution at 4 &#176;C for up to a week.</li>
</ol>
4. Fc Receptor-mediated Phagocytosis
<ol>
	<li>Aspirate the drug or medium supernatant from the 8-chamber slide and add 400 &#181;L of the 1.25% v/v R2R2 mixture. Incubate at 37 &#176;C for 2 h.</li>
	<li>After 2 h of incubation, remove the chambers using the manufacturer&#39;s adaptors. Dab off excess R2R2 on a paper towel. Make sure that the slide does not dry out.</li>
	<li>Fill a 100-mL beaker with PBS. Submerge and wash the slide by slowly moving the slide back and forth (around 30-40 strokes) to remove the majority of the unphagocytosed R2R2.</li>
	<li>Remove the slide from the PBS. Dab off excess PBS using a paper towel or tissue and air-dry the slide.</li>
	<li>Fix the air-dried slide in 100% methanol for 45 s. Then, air-dry the fixed slide. 
	NOTE: Slides can be fixed using another method that is more compatible with downstream staining, such as the Gr&#252;nwald-Giemsa stain21 or the Wright-Giemsa stain.</li>
	<li>Mount the slide using an in-house-made Elvanol mounting medium (or another commercially available mounting medium) and add coverslips. 
	NOTE: Elvanol mounting medium is composed of 15% w/v polyvinyl alcohol resin and 30% v/v glycerin in PBS. The mixture is heated until all the resin has dissolved and the glycerin is homogenously mixed. This can be replaced with other commercially available mounting medium.</li>
	<li>Allow the mount to dry overnight before quantification.</li>
</ol>
5. Quantification of Phagocytosis
<ol>
	<li>Using a phase-contrast microscope and a 40X objective lens, manually quantify the amount of phagocytic events by counting at least 200 monocytes and the number of phagocytosed R2R2 within these monocytes. Have one counter in each hand to simultaneously quantify the number of monocytes and the number of phagocytosed R2R2.</li>
	<li>Obtain the average phagocytic index by dividing the number of phagocytosed R2R2 by the number of monocytes and multiplying by 100. Express the data as the mean (average phagocytic index) &#177; the standard error of the mean (SEM).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>RPMI 1640</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>8-chamber slides</td>
			<td>Lab-Tek-ll</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>R2R2 (cDE/cDE) red blood cells</td>
			<td></td>
			<td></td>
			<td>Commercially available (e.g., http:// www.bio-rad.com/en-ca/product/ reagent-red-blood-cells)</td>
		</tr>
		<tr>
			<td>Polyclonal anti-D from human serum</td>
			<td>Gamma Biologics</td>
			<td>DIN 02247724</td>
			<td>Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin</td>
		</tr>
		<tr>
			<td>100% methanol</td>
			<td>Caledon</td>
			<td>6700-1-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>VWR</td>
			<td>48366 067</td>
			<td></td>
		</tr>
	</tbody>
</table>

an assay for studying fc&#947; receptor-driven phagocytosis in monocyte monolayers

Source: Tong, T. N., et al. <a href="https://app.jove.com/v/55039">Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis.</a> J. Vis. Exp. (2017)
This video showcases a monocyte monolayer assay utilizing primary monocytes from peripheral blood to assess Fc&#947; receptor-mediated phagocytosis with poly D antibody-opsonized R2R2 red blood cells.

An Assay for Studying Fc&#947; Receptor-Driven Phagocytosis in Monocyte Monolayers

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Peripheral Blood Mononuclear Cell ...

Begin with red blood cells, or RBCs.
Add polyclonal antibodies specific to RBC surface antigens.
Incubate. The antibodies opsonize RBCs by coating them through specific binding to surface antigens.
Centrifuge and remove the unbound antibodies.
Add the resuspended antibody-opsonized RBCs to a multi-chambered slide containing monocyte monolayers. Incubate.
The surface Fc gamma receptors on monocytes bind to the Fc region of polyclonal antibodies bound to RBCs.&#160;
This binding causes Fc receptor clustering around the contact area and triggers intracellular signaling, resulting in actin cytoskeleton reorganization in monocytes.
This cytoskeleton rearrangement induces pseudopod extensions and creates a phagocytic cup around opsonized RBCs.
The phagocytic cup progressively encloses opsonized RBCs, leading to their complete engulfment within a membrane-bound phagosome.
Post-incubation, wash the slide with buffer to remove non-phagocytosed RBCs. Fix the cells.
Mount the slide using mounting media.
Perform phase-contrast microscopy to visualize and quantify the phagocytosed RBCs within monocytes.

an-assay-for-studying-fc-receptor-driven-phagocytosis-monocyte

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

69 Views. Source: Tong, T. N., et al. Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis. J. Vis. Exp. (2017) This video showcases a monocyte monolayer assay utilizing primary monocytes from peripheral blood to assess Fcγ receptor-mediated phagocytosis with poly D antibody-opsonized R2R2 red blood cells. 

Video: An Assay for Studying Fcγ Receptor-Driven Phagocytosis in Monocyte Monolayers - Concept

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

JoVE Journal

Immunology and Infection

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 ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

An Assay for Studying Fcγ Receptor-Driven Phagocytosis in Monocyte Monolayers

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