eoe sub articles

EoE Sub Articles

1. Coupling of Magnetic Nanoparticles (MNPs)
<ol>
	<li>Use commercial coupling procedure and reagents to couple MNPs with antibodies (Abs) (typically 1 mg). Iron oxide nanoparticles with carboxylic acid as reactive groups are used.
	<ol>
		<li>If antibodies are in a volume higher than 0.5 ml, concentrate using a 100K concentrator. Spin at 2,000 x g for 3-5 min.</li>
	</ol>
	</li>
	<li>At the end of the procedure, after adding 10 &#181;l of the quenching buffer, transfer MNPs to a 12 mm x 75 mm tube and add 3 ml wash/storage buffer. Place the tube in the center hole of a magnetic separator and leave overnight at 4 &#176;C.</li>
	<li>Verify that MNPs have all collected to the side of the tube, and then carefully pipet out all liquid. Add 3 ml of fresh wash/storage buffer, and place back in the magnet.</li>
	<li>After several hr, check if MNPs are collected to the side of the tube and then pipet off the liquid. Use 2 ml of wash/storage buffer to resuspend the MNPs and store at 4 &#176;C.</li>
	<li>Verify that Abs are coupled to MNPs by labeling them with a relevant fluorescent Fab antibody fragment (e.g.&#160;goat anti-mouse if using a mouse monoclonal Ab for capture as described in section 2) and run on a flow cytometer.</li>
</ol>
2. Labeling Antibodies Coupled to MNPs
<ol>
	<li>Combine 60 &#181;l (3.9 x 1012&#160;particles) of MNPs (per condition) and 5 &#181;l (of a 200 &#181;g/ml commercial solution) labeled Fab fragments, specific for the isotype of the capture Ab in a 1.5 ml microcentrifuge tube.</li>
	<li>Incubate at room temperature (RT) for 30 min with continuous mixing.</li>
	<li>Pre-wet a 100K concentrator with 300 &#181;l of phosphate buffered saline (PBS) and spin in a microcentrifuge at 1,500 x g for 5 min.</li>
	<li>Purify labeled complexes on 100K column. Spin the mixture from step 2.2 in a microcentrifuge at 1,500 x g for 5 min, wash with 200 &#181;l PBS, and recover in the initial volume.</li>
</ol>
3. Use of Labeled Ab-MNP Complexes to Capture Particles of Interest (Extracellular vesicles, EVs)
<ol>
	<li>Incubate pre-labeled MNPs 60 &#181;l (3.9 x 1012&#160;particles) with EV preparation (100 &#181;l).
	<ol>
		<li>Prepare EV preparations using various methods. Here, purify EVs on sucrose gradients, collect them from platelet-poor plasma using exosome precipitation reagents or isolate directly from plasma.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: Optimal ratios need to be determined for each experiment and are dependent on the concentration of EV in the preparation. MNP-Ab fraction should be ~106&#160;in excess compared to concentration of EV fraction.</li>
	</ol>
	</li>
	<li>Incubate 1 hr at 4 &#176;C for EVs.</li>
	<li>Add 2.5% mouse IgG/ human IgG to block Fc binding, gently vortex, incubate 3-5 min at RT.</li>
	<li>Add manufacturer recommended or titered concentrations of each detection Abs and incubate additional 20 min at RT.</li>
</ol>
4. Separation of the MNP-captured EVs from Unbound Antibodies Using Magnetic Columns
<ol>
	<li>Prepare magnetic separation column for use by placing it in a separator magnet.</li>
	<li>Pre-wet the column with 500 &#181;l of wash buffer (2 mM Ethylenediaminetetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA) in PBS). Allow the wash buffer to flow through the column.</li>
	<li>Add the MNP-EV complexes to the column and allow all liquid to flow through. Add 500 &#181;l of wash buffer to the column, allowing the whole volume to pass through.</li>
	<li>Repeat washing with wash buffer 2 more times.</li>
	<li>Remove the column from magnet and place in 12 mm x 75 mm tube for collection of the retained MNP-EV complexes. Let the column stand in the tube off the magnet for 3 min. Add 200 &#181;l of PBS and let the beads flow down by gravity, add another 200 &#181;l of PBS, and fix with 200 &#181;l 4% paraformaldehyde after elution. 
	NOTE: Paraformaldehyde is a suspected carcinogen and should be handled in a ventilated hood and gloves should be worn.</li>
	<li>To quantify single EVs use volumetric settings on High Throughput Sampler (HTS) or just prior to acquisition add well-mixed flow cytometry count beads.</li>
</ol>
5. Analysis of MNPs-captured EVs with a Flow Cytometer
<ol>
	<li>Warm up flow cytometer for 30 min.</li>
	<li>Run quality control beads.</li>
	<li>Set threshold on fluorescence of either MNPs or labeled EVs. Use 0.22 &#181;m filtered PBS for threshold to decrease the background &#34;noise&#34;. Set the threshold by adjusting the PMT voltage at the level where filtered PBS gives no, or very few, events.</li>
	<li>Run samples on low. (Use an additional 0.04 &#181;m inline filter for sheath fluid placed in series behind the standard 0.2 &#181;m sheath filter to further eliminate false events).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxyl Magnetic Iron Oxide Nanoparticles Conjugation Kits</td>
			<td>Ocean Nanotech</td>
			<td>ICK-15-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td>Millipore</td>
			<td>UFC810024</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Round Bottom PP Test Tube, without Cap</td>
			<td>Falcon</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Quality Biological&#160;</td>
			<td>351-068-131</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMAG-01 magnetic separator&#160;</td>
			<td>Ocean Nanotech</td>
			<td>SuperMAG-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Zenon R-Phycoerythrin Mouse IgG1&#160;Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Z25055</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosep Centrifugal Devices with Omega Membrane 100k</td>
			<td>Pall Corp</td>
			<td>OD100C34</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5M</td>
			<td>Corning Cellgro</td>
			<td>46-034-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution</td>
			<td>Sigma-Aldrich</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, EM Grade, Purified</td>
			<td>EMS</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>123count eBeads&#160;</td>
			<td>eBioscience</td>
			<td>01-1234-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoCount beads</td>
			<td>Dako</td>
			<td>S2366</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuCheck count beads</td>
			<td>Invitrogen</td>
			<td>PCB100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytometer Setup &#38; Tracking Beads Kit (CST)</td>
			<td>BD Bioscience</td>
			<td>642412</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;MACS column&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-701</td>
			<td></td>
		</tr>
		<tr>
			<td>OctoMACS Separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>ThermoFisher</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flow cytometer</td>
			<td>BD Bioscience</td>
			<td>none</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo software</td>
			<td>Flowjo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunocapture and flow cytometry of extracellular vesicles for antigenic characterization

Source: Arakelyan, A. et al., <a href="https://app.jove.com/v/55020">Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the immunocapture of extracellular vesicles using fluorescent-labeled capture antibody-magnetic nanoparticle conjugates. The process involves complex formation, staining with fluorescently labeled detection antibodies, magnetic separation, and flow cytometric analysis for antigenic characterization.

Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

1. Coupling of Magnetic Nanoparticles (MNPs)

	Use commercial coupling procedure and reagents to couple MNPs with antibodies (Abs) (typically 1 mg). Iron ...

Begin with a tube carrying cell-derived membrane-bound particles termed extracellular vesicles, or EVs, that exhibit unique antigenic compositions.
Introduce magnetic nanoparticles, MNPs, containing capture antibodies conjugated with fluorescent-labeled Fab fragments.
Incubate to facilitate the interaction between the MNPs with specific antigens of EVs, forming MNP-EV complexes.
Add immunoglobulin-G to block Fc binding sites, then introduce fluorescently labeled detection antibodies to stain other antigens on the captured particles.
Load this mixture onto a pre-washed magnetic column placed in a magnetic separator.
The magnetic field retains MNP-captured particles within the column. Exchange the buffer to remove unbound antibodies.
Retrieve the column from the magnetic separator, elute MNP-captured particles with a buffer, and fix them to preserve their structural integrity.
Analyze the immunocaptured particles by flow cytometry; distinct fluorescence signals from stained antigens correlate with unique antigenic characteristics of EVs.

immunocapture-flow-cytometry-extracellular-vesicles-for-antigenic

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

80 Views. Source: Arakelyan, A. et al., Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles. J. Vis. Exp. (2017) This video demonstrates the immunocapture of extracellular vesicles using fluorescent-labeled capture antibody-magnetic nanoparticle conjugates. The process involves complex formation, staining with fluorescently labeled detection antibodies, magnetic separation, and flow cytometric analysis for antigenic characterization. 

Video: Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization - Concept

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

JoVE Journal

Cancer Research

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Immunology and Infection

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

Transcript

Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media