an opsono-adherence assay for assessing the opsonization of encapsulated bacteria by anti-capsule antibodies

An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies

Wash the inactivated bacterial stock three times in PBS to remove fixative. Add FITC solution at a final concentration of 75 micrograms per milliliter. Slowly, agitate the mixture for 18 to 24 hours at four degrees Celsius. Wash the bacteria as before to remove excess FITC.
After the final wash, resuspend the bacilli in PBS in the same start volume. Aliquot and store bacilli in -20 degrees Celsius until use. Heat-inactivate a small volume of test sera from non-human primates at 56 degrees Celsius for 30 minutes, in a heat block or PCR machine. Thaw and wash FITC-labeled bacteria with PBS two times by pelleting at 15,000 times g for three to five minutes. Resuspend in PBS in the same start volume.
In a 96-well round-bottom plate, serially dilute the test sera in cell medium. In another 96-well round-bottom plate, add 10 microliters of diluted test sera into each well. Then, add 10 microliters of freshly reconstituted baby rabbit complement.
To plate number three, add the appropriate volume of FITC-labeled bacilli for a multiplicity of infection of 20, for example, at a volume containing 50,000 times 20 bacilli for 50,000 cells. Top off each well in plate number three with the appropriate volume of cell medium to a total of 105 microliters. Incubate plate number three at 37 degrees Celsius for 30 minutes in the presence of 5% CO2 with humidity to opsonize bacilli.
Place plate number one which contains adherent cells on a 37 degrees Celsius heat block. Use a multichannel pipetter to remove spent medium from wells and quickly replace with 100 microliters of opsonized bacteria from plate number three ensuring no bubbles are generated in the wells during pipetting. To not disturb the cell monolayer, add volume to the sides of the wells.
Incubate plate number one at 37 degrees Celsius with 5% CO2 and humidity for 30 minutes for adherence. Wash each well five times with 150 microliters of PBS on top of a 37 degrees Celsius heat block to remove unattached bacilli. Pipette to the side of the wells to ensure that the cells are not accidentally dispersed during washing. Add 150 microliters of 4% paraformaldehyde to each well, and fix the cells for one hour.
After washing the cells twice with PBS, mix two microliters of high-content screening orange cell stain in 10 milliliters of PBS. Then, add 150 microliters of this staining solution to fixed cells, and incubate for 30 minutes at ambient temperature. Place plate number one on the stage and focus on the cells using bright-field or phase contrast.
In the Acquisition tab, select 96-Well format as the plate setting then select Channel settings. Next, select Setting to Tile Mode. Indicate the number of images to be acquired. Change the acquisition mode to black and white or monochrome, and set the binning to at least 2 by 2. Turn on the autofocus or focusing module. Indicate how often the autofocusing function is to be performed.
Next, turn on the z-stack function if available indicate the number of z-stacks that will capture the thickness of the cell monolayer and adherent bacilli. Designate a file folder to save the images to. Now, run the automated imaging program. Perform data collection and analysis, as described in the text protocol.

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1. Bacterial Culture and Preparations
NOTE: The bacterial species used in this protocol is an encapsulated virulent strain, B. anthracis Ames. All manipulations with this species require appropriate biosafety and security clearances and must be performed in a Class II or Class III biological safety cabinet located in a BSL-3 laboratory. Follow institutional operating procedures for BSL-3 work and for the use of personal protective equipment when handling bacteria.
<ol>
	<li>Prepare Brain Heart Infusion (BHI) broth by dissolving 37 g BHI powder in 1 L water and autoclaving at 121 &#176;C for 15 min. Store broth at ambient temperature.</li>
	<li>Prepare 8% sodium bicarbonate solution in water and sterilize using a 0.20 &#181;m filter syringe. Store solution at 4 &#176;C and use within 1 day.</li>
	<li>Mix 8 g Nutrient Broth, 3 g yeast extract, and 15 g agar in 1 L water to prepare NBY agar plates. Autoclave at 121 &#176;C for 15 min. Pour into Petri dishes and allow to solidify. Store plates at 4 &#176;C. 
	NOTE: NBY agar plates may be made with or without sodium bicarbonate. The addition of sodium bicarbonate to broth and agar plates induces the growth of mucoid colonies consisting of encapsulated bacilli. The final concentration of sodium bicarbonate in both liquid and solid media is 0.8%.</li>
	<li>Prepare culture broth for B. anthracis by mixing 50% autoclaved BHI broth, 40% FBS, and 10% sodium bicarbonate solution (v/v). 
	NOTE: This broth is formulated to produce short chains of encapsulated bacilli.</li>
	<li>Prepare a master plate of B. anthracis by streaking it for isolation on an NBY agar plate from a spore stock one day before culturing in broth. Incubate at 37 &#176;C.</li>
	<li>Inoculate 30 mL of culture broth in a vented 250 mL flask with several colonies of B. anthracis. 
	NOTE: A specific volume-to-surface ratio of broth, as specified here, is needed to produce maximally encapsulated bacilli.</li>
	<li>Shake the broth culture at 125 rpm, 37 &#176;C with 20% CO2 and humidity for 18&#8211;24 h. 
	NOTE: Growing B. anthracis at temperatures greater than 37 &#176;C may inhibit capsule production.</li>
	<li>Use negative staining with India ink to ensure sufficient encapsulation and that bacilli are in short chains (1&#8211;5 bacilli/chain) before fixation (see section 2).</li>
	<li>To determine colony forming units (CFU), serially dilute 100 &#181;L of culture 1:10 in Phosphate Buffered Saline (PBS) solution and plate culture dilutions on sheep blood agar plates. Incubate for 12&#8211;18 h at 37 &#176;C. Count CFUs and determine the number of bacteria per mL of culture. 
	NOTE: Counting can also be performed using a hemocytometer under the microscope once the bacteria have been fixed. However, this is not recommended because the bacilli are difficult to see under low magnification.</li>
	<li>Add 16% paraformaldehyde (PF) to the entire volume of bacterial culture at a final concentration of 4% PF to fix the bacilli. Slowly agitate on a shaker at ambient temperature for 7 days. 
	NOTE: Seven days of incubation in 4% PF is based on the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) institutional standard operating procedure for fixing vegetative bacilli.</li>
	<li>To remove the fixative and test for sterility, thrice wash 4 mL (10% volume) of fixed bacterial suspension in a 50 mL conical tube by centrifugation at &#8805;3000 x g for 45 min and using a 30 mL/wash with PBS. Store the remaining sample in 4% PF at 4 &#176;C. 
	NOTE: After pelleting, the bacterial pellet is fluffy due to the presence of a capsule. Take care not to disturb the pellet during washing. It is necessary to remove all fixative so that it does not interfere with the viability testing. If necessary, increase the number of washes.</li>
	<li>For viability testing, inoculate 40 mL of a bacterial growth medium (e.g. BHI broth) with 4 mL of washed suspension (&#8804; 10% of broth volume) and incubate at 37 &#176;C for 7 days. Check optical density at 600 nm before and after 7 days to measure turbidity.</li>
	<li>After 7 days of incubation in broth culture, plate &#8805;100 &#181;L of broth onto an agar plate and incubate for another 7 days. If no increase in turbidity and no growth on plates are seen, the original culture is considered sterile and may be transferred to a BSL-2 laboratory. 
	NOTE: The Federal Select Agent Program mandates that inactivated B. anthracis can only be transferred from BSL-3 laboratories after demonstrating complete sterility of the sample. Viability testing for inactivated B. anthracis consists of culturing 10% of the sample in broth for 7 days followed by culturing on a solid medium for another 7 days. Follow institutional guidelines to receive transfer approval once sterility is established. &#160;&#160;&#160;&#160;&#160;&#160; &#160;</li>
</ol>
2. Negative staining and light microscopy to observe encapsulation and bacilli chains
<ol>
	<li>Mix 5 &#181;L bacterial suspension with 2 &#181;L of India ink on a microscope slide. Place a 1 or 1.5 &#181;m thick cover glass on top of the suspension without creating bubbles. 
	NOTE: Nail polish may be used to seal the cover glass onto the slide.</li>
	<li>Observe the bacterial suspension using a 40x to 100x oil objective lens on a light microscope. Ensure that the bacilli are encapsulated by observing a zone of clearance (capsule excludes India ink) surrounding each bacterium and that the bacilli chains are short.</li>
</ol>
3. FITC-labeling of bacteria
<ol>
	<li>Dissolve fluorescein isothiocyanate (FITC) in dimethyl sulfoxide at a concentration of 2 mg/mL.</li>
	<li>Measure the volume of inactivated bacterial stock.</li>
	<li>Wash the stock 3x in PBS to remove the fixative.</li>
	<li>Add FITC solution at a final concentration of 75 &#181;g/mL. Slowly agitate the mixture for 18&#8211;24 h at 4 &#176;C.</li>
	<li>Thrice wash the bacteria by pelleting at &#8805;3000 x g, 45 min, and using 30 mL PBS/wash to remove excess FITC. Ensure that the fluffy pellet is not disturbed during washing. Resuspend the bacilli in PBS at the same start volume.</li>
	<li>Aliquot and store the bacilli in microtubes at -20 &#176;C until use. Do not freeze/thaw bacilli multiple times as the bacilli may lose their capsule.</li>
</ol>
4. Bacterial Opsonization
<ol>
	<li>Heat inactivate a small volume of test sera (from non-human primates, NHPs) at 56 &#176;C for 30 min in a heat block.</li>
	<li>Thaw and wash FITC-labelled bacteria with PBS 2x by pelleting at 15000 x g for 3&#8211;5 min. Resuspend in PBS at the same start volume.</li>
	<li>In a 96-well round bottom plate (plate #2), serially dilute test sera in cell medium. In another 96-well round bottom plate (plate #3), add 10 &#181;L of diluted test sera into each well. 
	NOTE: In every plate, PBS and normal monkey sera were included in place of diluted test sera as negative controls. We also chose one test serum as a positive control to generate a standard curve to normalize all assays done on different days. The positive control test serum was arbitrarily chosen because it was plentiful.</li>
	<li>To plate #3, add 10 &#181;L freshly reconstituted baby rabbit complement. 
	NOTE: Once reconstituted, discard the remaining baby rabbit complement; do not refreeze and thaw.</li>
	<li>To plate #3, add the appropriate volume of FITC-labeled bacilli for a multiplicity of infection of 1:20. For example, add the volume that contains 5 x 104 x 20 bacilli for 5 x 104 cells. Top off each well in plate #3 with the appropriate volume of cell medium to total 105 &#181;L. 
	NOTE: Plate #1, which contains cells, receives 100 &#181;L of opsonized bacteria with the remaining 5 &#181;L as the void volume. We tested each dilution of the test sera in duplicate.</li>
	<li>Incubate plate #3 at 37 &#176;C for 30 min in the presence of 5% CO2 with humidity to opsonize bacilli.</li>
</ol>
5.Adherence Assay and Fluorescent Labeling of Mammalian cells
<ol>
	<li>Maintain all reagents at 37 &#176;C before use.</li>
	<li>Place plate #1, which contains adherent cells, on a 37 &#176;C heat block.</li>
	<li>Use a multichannel pipettor to remove the spent medium from wells and quickly replace it with 100 &#181;L of opsonized bacteria from plate #3. Ensure no bubbles have been generated in the wells during pipetting. Incubate plate #1 at 37 &#176;C with 5% CO2 and humidity for 30 min for adherence.</li>
	<li>Wash each well 5x with 150 &#181;L PBS on top of a 37 &#176;C heat block to remove unattached bacilli. 
	NOTE: Care must be taken to ensure that the cells are not accidentally dispersed during washing.</li>
	<li>Dilute 16% PF with PBS 1:4 to make 4% PF. Fix cells with 4% PF for 1 h by adding 150 &#181;L to each well. Wash cells 2x with PBS.</li>
	<li>Mix 2 &#181;L HCS (high-content screening) Orange Cell Stain in 10 mL PBS. Add 150 &#181;L of this staining solution to fixed cells and incubate for 30 min at ambient temperature.</li>
	<li>Wash wells 2x with PBS.</li>
	<li>Store at 4 &#176;C until microscopy.</li>
</ol>
6. Microscopy
NOTE: In general, a high-throughput automated fluorescence microscope will facilitate imaging for the OAA. If an automated system is not available, fluorescent images of adherent bacilli can be taken manually. In this study, adherent bacilli and cell monolayers were imaged using the Zeiss 700 laser scanning microscopy system with specialized equipment; our system was composed of the Zeiss Axio Observer Z1 inverted microscope equipped with an automated stage, the Definite Focus module, 40 x NA 0.6 objective, Axio Cam HRc camera and Zen 2012 (Blue Edition) software. The images acquired are widefield images, not confocal images. The following protocol is intended as a basic microscope setup that is applicable to a variety of automated microscopy systems.
<ol>
	<li>Incubate plate #1 at ambient temperature for 30 min. Turn on the microscope and related equipment. 
	NOTE: Acclimation at ambient temperature prevents condensation from forming on the glass plate during imaging. In addition, it prevents defocusing due to temperature shifts.</li>
	<li>Place the plate on the stage and focus on the cells using a bright field or phase contrast.</li>
	<li>Select 96 well format as the plate setting. Select channel settings. 
	NOTE: FITC&#39;s peak excitation and emission wavelength are 495/519 nm; the HCS orange cell stains are 556 and 572 nm. Other fluorophores may be used depending on the filters that are available on the microscope.</li>
	<li>Select setting to Tile mode. Indicate the number of images to be acquired. 
	NOTE: The tiling function allows the capture of multiple locations in a sample well and can be set to acquire an image in a tiling or a random format. We imaged 10 random areas per well.</li>
	<li>Change acquisition mode to &#34;black &#38; white&#34; or &#34;monochrome&#34; and binning &#8805; 2x2. 
	NOTE: Setting the binning from 1x1 to 2x2 or 4x4 would increase microscopy speed but would also result in a lower resolution of the image. For OAA, it is sufficient to use these settings as the assay enumerates both the macrophages and the adherent bacteria.</li>
	<li>Turn on the autofocus or focusing module. Indicate how often the autofocusing function is to be performed. Turn on the Z stack function, if available. Indicate the number of Z stacks that will capture the thickness of the cell monolayer and adherent bacilli. 
	NOTE: The number of Z stacks chosen will increase microscopy time but will ensure an image with the correct focus is taken at each Z stack.</li>
	<li>Designate a file folder to save images to. Run the automated imaging program.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.20 &#181;m syringe filter (25mm, regenerated cellulose)</td>
			<td>Corning, Corning, NY</td>
			<td>431222</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe (Luer-Lock tip)</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>15&#181; 96 well black plates (plate #1 for imaging)</td>
			<td>In Vitro Scientific, Sunnyvale, CA</td>
			<td>P96-1-N</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm sq. tissue culture treated flask</td>
			<td>Corning, Corning, NY</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar (powder)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Baby Rabbit Complement</td>
			<td>Cedarlane Labs, Burlington, NC</td>
			<td>CL3441</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>BD, Sparks, MD</td>
			<td>288620</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion (BHI)</td>
			<td>BD, Sparks, MD</td>
			<td>211059</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar (TSA with Sheep Blood) plates</td>
			<td>Remel, Lenexa, KS</td>
			<td>R01198</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sarstedt, Newton, NC</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 96 well cell culture plates (plates #2 &#38; 3 for dilutions)</td>
			<td>Corning, Corning, NY</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Electron Microscopy Science, Hatfield, PA</td>
			<td>72200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Nutrient Broth</td>
			<td>BD, Sparks, MD</td>
			<td>234000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham, MA</td>
			<td>11965-092</td>
			<td>Contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red</td>
		</tr>
		<tr>
			<td>EVOS FL Auto Cell Imaging System (fluorescence microscope)</td>
			<td>Life Technologies, Thermo Fisher Scientific, Waltham, MA</td>
			<td>AMAFD1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SH30071.03</td>
			<td>Not gamma irradiated, not heat inactivated</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>F143</td>
			<td></td>
		</tr>
		<tr>
			<td>HCS Cell Mask Orange Cell Stain</td>
			<td>Invitrogen, Thermo Fisher Scientific, Waltham, MA</td>
			<td>H32713</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer (Improved Neubauer)</td>
			<td>Hausser Scientific, Horsham, PA</td>
			<td>3900</td>
			<td></td>
		</tr>
		<tr>
			<td>India Ink solution</td>
			<td>BD, Sparks, MD</td>
			<td>261194</td>
			<td></td>
		</tr>
		<tr>
			<td>L- glutamine (200 mM)</td>
			<td>Gibco, Thermo Fisher Scientific, Waltham MA</td>
			<td>25030081</td>
			<td>Supplement medium with additional 2mM L-glutamine</td>
		</tr>
		<tr>
			<td>Nikon Eclipse TE2000-U (inverted compound microscope)</td>
			<td>Nikon Instruments, Melville, NY</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Calcium or Magnesium</td>
			<td>Lonza, Walkersville, MD</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100x</td>
			<td>Hyclone, GE Healthcare Life Sciences, South Logan, UT</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100 x 15 mm)</td>
			<td>Falcon, Corning, Durham, NC</td>
			<td>351029</td>
			<td>For agar plates</td>
		</tr>
		<tr>
			<td>RAW 264.7 macrophage cell line (Tib47)</td>
			<td>ATCC, Manassas, VA</td>
			<td>ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR, Radnor, PA</td>
			<td>16004-422</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 700 Laser Scanning Microscopy (confocal microscope)</td>
			<td>Carl Zeiss Microimaging, Thornwood, NY</td>
			<td>4.109E+15</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Chua, J., et al. <a href="https://app.jove.com/v/60873">Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens.</a> J. Vis. Exp. (2020)
This video demonstrates an opsono-adherence assay for examining the interaction between opsonized bacteria and immune cells. Fluorescently labeled bacteria, treated with test serum containing anti-capsule antibodies, are incubated with macrophages. After staining the macrophages with fluorescence, the attached bacteria on the macrophage surface are observed under a microscope for visualization.

1. Bacterial Culture and Preparations
NOTE: The bacterial species used in this protocol is an encapsulated virulent strain, B. anthracis Ames. All ...

Take a suspension of chemically-killed, fluorescently labeled Bacillus anthracis.
The bacterial capsule, mainly composed of poly-gamma-glutamic acid, provides protection against phagocytosis.
Take non-human primate sera, heat-inactivated to destroy complement proteins. These sera, isolated from animals immunized with a bacterial-capsule-based vaccine, contain anti-capsule antibodies.
Add the chemically killed bacterial suspension to the sera. The antibodies specifically bind to the bacterial capsules, facilitating bacterial opsonization and marking them for elimination by phagocytic cells. Add standard complement proteins to enhance opsonization.
Transfer the opsonized bacteria to adherent macrophages &#8212; phagocytic cells. Incubate briefly.
The antibodies and complement proteins on the bacteria bind to macrophage Fc receptors and complement receptors, respectively, promoting bacterial adherence.
Remove media. Wash with buffer to remove non-adhered bacteria.
Fix the cells to preserve cellular integrity.
Use a fluorescent stain to label the macrophages.
Using a fluorescence microscope, observe fluorescent bacteria adhered to differently-fluorescent macrophages, confirming effective bacterial opsonization by anti-capsule antibodies.

25 vistas. Un ensayo de opsonoadherencia para evaluar la opsonización de bacterias encapsuladas por anticuerpos anticápsulas

Video: Un ensayo de opsonoadherencia para evaluar la opsonización de bacterias encapsuladas por anticuerpos anticápsulas - Experimento

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

El uso de una monocapa de monocitos Ensayo para evaluar la fagocitosis mediada por el receptor Fc gamma

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Investigación

JoVE Journal

Inmunología e Infección

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.

Proyección de imagen 3D Time-lapse de la fagocitosis por macrófagos de ratón

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

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Medición de anticuerpos que inducen la fagocitosis adquirida naturalmente a los parásitos plasmodium falciparum mediante un ensayo basado en citometría de flujo

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies

Transcripción

An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies