This work of AA, SZ, WF, J-C.G, and LM was supported by the NICHD Intramural Program. The work of MV was supported by the Russian Federation Government grant #14.B25.31.0016.

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. 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 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 (Viruses or EVs)
<ol>
	<li>Incubate pre-labeled MNPs 60 &#181;l (3.9 x 1012 particles) with HIV (60 &#181;l) or 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 8. 
		NOTE: Optimal ratios need to be determined for each experiment and are dependent on concentration of virus/EV in the preparation. MNP-Ab fraction should be ~106 in excess compared to concentration of virus/EV fraction.</li>
	</ol>
	</li>
	<li>Incubate 40 min at 37 &#176;C with gentle mixing for viruses, or 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 Virions (or 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-virus/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-virus/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 virions/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 Viruses/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 viruses/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>
6. Evaluation of the Capture Efficiency
<ol>
	<li>Capture fluorescently labeled EVs or HIV with MNP-Abs as described in 3.1, 3.2. (EVs can be labeled either with a membrane dye or through their cargo 15; HIV can be labeled either through gene-encoded dye 16, or with a membrane dye 12,17).</li>
	<li>Prepare magnetic separation column as described in (4.1-4.2).</li>
	<li>Add the MNP-virus/MNP-EV complexes to the column and allow all liquid to flow through (flow through fraction). Collect the flow through fraction in tube.</li>
	<li>Proceed with fraction retained on the column as described in (4.4-4.5).</li>
	<li>Repeat capture procedure on the flow through fraction from 6.3 with subsequent separation (4.1-4.5).</li>
	<li>Analyze the retained fractions from first capture and second capture by running them on cytometer that is set on triggering on fluorescent label of EVs or HIV. To evaluate efficiency, compare the number of events in both fractions.</li>
</ol>
7. Adaptation of the Protocol for Specific Tasks
NOTE: While the protocol can be applied to analysis of HIV and EVs as described above, for analysis of DENV the following modifications should be introduced:
<ol>
	<li>Incubate virions with 1 &#181;M DiI in the dark for 30 min at RT. After incubation purify stained virus by centrifugation in density gradient medium (10%, 20%, 25% and 35%) at 240,000 x g for 1.5 hr at 4 &#176;C with no brake. Collect fraction between 20% and 25% density gradient medium layers 14.</li>
	<li>Incubate DiI labeled DENV 1 x 107 (60 &#181;l) with manufacturer recommended or titered concentrations of detection Ab in the presence of 2.5% mouse IgG for 20 min at RT.</li>
	<li>Incubate mixture with pre-labeled 60 &#181;l (3.9 x 1012 particles) MNP-capture Ab complexes for 40 min at 37 &#176;C with gentle mixing.</li>
	<li>Separate resultant complexes from unbound Abs on magnetic columns as described above and analyze on flow cytometer.</li>
</ol>

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F., Cantin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acquisition+of+host-encoded+proteins+by+nascent+HIV-1.">The acquisition of host-encoded proteins by nascent HIV-1.</a> Immunol Today. 19 (8), 346-351 (1998).</li><li>Santos, S., Obukhov, Y., Nekhai, S., Bukrinsky, M., Iordanskiy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-producing+cells+determine+the+host+protein+profiles+of+HIV-1+virion+cores.">Virus-producing cells determine the host protein profiles of HIV-1 virion cores.</a> Retrovirology. 9, 65 (2012).</li><li>Wolf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+and+significance+of+platelet+products+in+human+plasma.">The nature and significance of platelet products in human plasma.</a> Br J Haematol. 13 (3), 269-288 (1967).</li><li>Piccin, A., Murphy, W. G., Smith, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microparticles:+pathophysiology+and+clinical+implications.">Circulating microparticles: pathophysiology and clinical implications.</a> Blood Rev. 21 (3), 157-171 (2007).</li><li>Bernal-Mizrachi, L., 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=High+levels+of+circulating+endothelial+microparticles+in+patients+with+acute+coronary+syndromes.">High levels of circulating endothelial microparticles in patients with acute coronary syndromes.</a> Am Heart J. 145 (6), 962-970 (2003).</li><li>Rozmyslowicz, T., 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=Platelet-+and+megakaryocyte-derived+microparticles+transfer+CXCR4+receptor+to+CXCR4-null+cells+and+make+them+susceptible+to+infection+by+X4-HIV.">Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV.</a> Aids. 17 (1), 33-42 (2003).</li><li>Bhattarai, N., 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=GB+virus+C+particles+inhibit+T+cell+activation+via+envelope+E2+protein-mediated+inhibition+of+TCR+signaling.">GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of TCR signaling.</a> J Immunol. 190 (12), 6351-6359 (2013).</li><li>Arakelyan, A., Ivanova, O., Vasilieva, E., Grivel, J. C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+composition+of+single+nano-sized+extracellular+blood+vesicles.">Antigenic composition of single nano-sized extracellular blood vesicles.</a> Nanomedicine. 11 (3), 489-498 (2015).</li><li>Arakelyan, A., Fitzgerald, W., Margolis, L., Grivel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+flow+virometry+for+the+analysis+of+individual+virions.">Nanoparticle-based flow virometry for the analysis of individual virions.</a> J Clin Invest. 123 (9), 3716-3727 (2013).</li><li>Yang, H., Liang, W., He, N., Deng, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+labels+released+from+long+spacer+arm-functionalized+magnetic+particles:+a+novel+strategy+for+ultrasensitive+and+highly+selective+detection+of+pathogen+infections.">Chemiluminescent labels released from long spacer arm-functionalized magnetic particles: a novel strategy for ultrasensitive and highly selective detection of pathogen infections.</a> ACS Appl Mater Interfaces. 7 (1), 774-781 (2015).</li><li>Tang, Y., 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=Highly+sensitive+and+rapid+detection+of+Pseudomonas+aeruginosa+based+on+magnetic+enrichment+and+magnetic+separation.">Highly sensitive and rapid detection of Pseudomonas aeruginosa based on magnetic enrichment and magnetic separation.</a> Theranostics. 3 (2), 85-92 (2013).</li><li>Borlido, L., Azevedo, A. M., Roque, A. C., Aires-Barros, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+separations+in+biotechnology.">Magnetic separations in biotechnology.</a> Biotechnol Adv. 31 (8), 1374-1385 (2013).</li><li>Xi, Z., 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=Selection+of+HBsAg-Specific+DNA+Aptamers+Based+on+Carboxylated+Magnetic+Nanoparticles+and+Their+Application+in+the+Rapid+and+Simple+Detection+of+Hepatitis+B+Virus+Infection.">Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection.</a> ACS Appl Mater Interfaces. 7 (21), 11215-11223 (2015).</li><li>Zicari, 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=Evaluation+of+the+maturation+of+individual+Dengue+virions+with+flow+virometry.">Evaluation of the maturation of individual Dengue virions with flow virometry.</a> Virology. 488, 20-27 (2016).</li><li>Lai, C. P., 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=Visualization+and+tracking+of+tumour+extracellular+vesicle+delivery+and+RNA+translation+using+multiplexed+reporters.">Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.</a> Nat Commun. 6, 7029 (2015).</li><li>McDonald, 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=Visualization+of+the+intracellular+behavior+of+HIV+in+living+cells.">Visualization of the intracellular behavior of HIV in living cells.</a> J Cell Biol. 159 (3), 441-452 (2002).</li><li>Morcock, D. R., 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=Elimination+of+retroviral+infectivity+by+N-ethylmaleimide+with+preservation+of+functional+envelope+glycoproteins.">Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.</a> J Virol. 79 (3), 1533-1542 (2005).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical Flow Cytometry. , 101-223 (2005).</li></ol>

The authors declare no conflict of interest.

Conventional flow cytometers remain the major tool for analyzing cells based on their physical and chemical characteristics on an individual level 18. The basic principles of flow cytometry have not been changed since its development: a cell that passes through the laser beam and scatters light constitutes an event that triggers the recording of the fluorescent spectra emitted by cell-bound fluorescent antibodies. Smaller particles below 300 nm cannot be detected by side scatter of a cytometer as they scatter more light under larger angles at which most flow cytometer do not collect light 18. The technique described here, allows the identification and the analysis of multiple antigens on numerous individual viruses or EVs by conventional flow cytometers with fluorescence triggering.
The critical steps within the protocol are the coupling of antibodies to MNPs. Our experiments were performed using 15 nm iron oxide nanoparticles with carboxylic acid as a reactive group as described previously 9. According to the manufacturer, each 15 nm MNP can bind a maximum of 20 antibodies; we bind approximately 10 antibodies per 15 nm MNP. This estimate is based on the amount of antibodies before and after coupling and the number of beads/mL as measured by nanoparticle characterization and sizing instrumentation. In principle, coupling may cause aggregation of particles and is undesirable. To check that aggregation is not happening with our protocol, we verified this with nanoparticle characterization and sizing instrumentation. This instrument measures the hydrodynamic diameter for the particles, which includes the coating and antibody layers rather than the 15 nm core only. We found that the majority of coupled beads are located at a single peak (mean peak at 64.7 &#177;4.2 nm with 90% of all events below 73 &#177;7.3 nm). The mixing of Ab-MNP complexes with virions or EVs in the right proportion is important so that MNPs are in the concentration of several orders higher than EVs/virions. This is critical to ensure that single EVs/virions are analyzed. Avoid coincidences of the events by running cytometer at low flow. Ensure to trigger the acquisition on fluorescence and use volumetric controls to measure the concentration of the captured entities.
However, MNPs can aggregate EVs or virions by crosslinking or by the binding of two or more virions to the same nanoparticle. To check this, a test for aggregation as described in Figure 4 has to be performed. Even individually captured virions or EVs may simultaneously enter the laser beam of the flow cytometer. This would create false-positivity for two different antigens. To avoid this, the flow cytometer should be set as described and the preparations should be diluted. One can check whether coincidence is happening by using the same strategy as described in the previous point. Also, the lack of coincidence could be verified by analyzing serial dilutions of the preparation and proving that the number of events decreases linearly with the dilution while the mean fluorescence intensity of a fluorescently stained antigen remains constant in all dilutions (Figure 5). Another issue is that too few viruses/EVs can be captured. This can be due to the true scarcity of viruses/EVs that carry antigens through which they are captured by specific MNPs. Alternatively, the efficiency of capture is low due to various reasons (e.g., inefficient coupling of antibodies to MNPs, deviation from the ratios of MNPs to virions/EVs developed for this protocol, etc.). To avoid the latter possibility, the efficiency of capture should be specifically evaluated. Typically, the efficiency should be about 90% as in Figure 6.
Different antibodies can interfere due to steric hindrance with each other or with the antibodies coupled to MNPs. To check that this did not happen a reverse capture protocol must be tested (as described in Section 7. Briefly, virions or EVs should be first stained with fluorescent detection Abs and then captured by Ab-MNP complexes with subsequent separation from free floating Abs on magnetic columns.
Flow virometry has significant advantages with respect to existing methods of analysis of the antigenic composition of virions or EVs. Routine biochemical methods report on the general presence of particular proteins in the preparations, but provide no information on the heterogeneity of the virions or EVs. This includes immunocapture of virions or EVs on large commercially available particles. Such particles are typically of several microns in diameter and each of them can capture hundreds or thousands of virions or EVs. Therefore, any subsequent analysis averages the properties of the virions/EVs captured by one particle. In contrast, flow virometry uses MNPs of 10-15 nm in diameter and with the described protocol at least 90% of events represent a single virion or single EV.
The main limitation is that the entire population of virions or EVs in the preparation might not be analyzed, but only those that are captured. Thus, the choice of the antigen through which virions or EVs are captured becomes critical. Also, unlike flow cytometry the number of different antigens (the number of fluorescent antibodies against different antigens) is limited by the small surface of virions or EVs. Finally, different antibodies may sterically interfere with each other again due to the small (compared to the cells) surface.
The current protocol can be adapted for analysis of any virus or EV of any origin, provided that the specific antibodies through which they can be captured are available. Analysis of antigens on individual virions permits researchers to address the pathogenesis of different fractions of viral populations. Furthermore, identification of individual EVs in plasma may make it possible to trace the origin of EVs to particular cells and to report about normal or pathological conditions of these cells and the organs in which they reside.

Cells in multicellular organisms have many individual features that may be either unique for a particular cell or at least belong to unique groups of cells. Even cultured cloned cells show individual characteristics as evidenced by their diverse morphology. Also, a cell&#39;s individuality is reflected in the products it secretes. In the case of viral infection, viral particles can carry proteins from the cells that produced them. For example, for HIV many host cell proteins are embedded in the viral envelope and viruses that are generated by different cells may carry these characteristic proteins 1,2 or &#34;cell signatures&#34;.
Even without infection, cells release into the surrounding media small extracellular vesicles (EVs). Biogenesis of these vesicles and their structure in many cases resemble that of viruses, especially retroviruses. In recent years it has become clear that EVs, which were initially considered to be &#34;platelet dust&#34; 3, constitute an important physiological system of cell-cell communication. Together with two other systems of intercellular communication, namely cell-cell contact interactions and released soluble molecules, EVs coordinate normal physiology and are altered in various pathologies 4,5 including viral infections 6,7.
Although both released viral particles and extracellular vesicles are highly diverse, they are characterized predominantly by bulk analysis in which their individual characteristics are lost. A method akin to flow cytometry, which phenotypes cells based on their different surface antigens, is needed to characterize individual viral and viral-like particles. Unfortunately, EVs or small virions cannot be analyzed using standard flow cytometry methods because they are too small to generate the light-scattering signal on which most cytometers rely for triggering. To circumvent this limitation, fluorescence triggering can be applied. However, if EVs or virions are stained with fluorescent antibodies it is difficult to distinguish them from free antibodies and their aggregates because of their similar fluorescence and comparable sizes.
Recently, we developed a nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers 8,9. We employ MNPs to immunocapture particles of interest, as has been described by other groups for diverse applications 10,11,12,13; however, our novel technique allows for the capture of individual specific targets followed by phenotyping of these captured particles by flow cytometry using fluorescence triggering rather than light scattering, without interference from free floating antibodies. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particle generated by cells in vivo and in vitro provided the availability of specific fluorescent antibodies against surface antigens. We have already applied this technique to study several biological problems.
In particular, we evaluated the distribution of host cell markers on individual HIV-1 particles 9, we studied the maturation of individual Dengue viruses (DENV) based on analysis of their surface proteins 14 and investigated EVs released into the bloodstream of healthy volunteers and patients with acute coronary syndrome (ACS). Although based on a similar principle, the application of the new technique required development of specific protocols that are described below.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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>5 ml 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&amp;nbsp;</td><td>351-068-131</td><td></td></tr><tr><td>SuperMAG-01 magnetic separator&amp;nbsp;</td><td>Ocean Nanotech</td><td>SuperMAG-01</td><td></td></tr><tr><td>Zenon R-Phycoerythrin Mouse IgG1&amp;nbsp;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 0.5 M</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>123 count eBeads&amp;nbsp;</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 &amp;amp; Tracking Beads Kit (CST)</td><td>BD Bioscience</td><td>642412</td><td></td></tr><tr><td>Vybrant DiI Cell-Labeling Solution</td><td>Thermo Fisher Scientific</td><td>V22885</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;micro;MACS column&amp;nbsp;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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>

flow virometry to analyze antigenic spectra of virions and extracellular vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Selective capture of cellular antigens by HIV-1 virions
With &#34;flow virometry&#34; it is now possible to visualize cellular antigens on individual viral particles. As an example, we focused on two cellular antigens carried by HIV-1, LFA-1 and HLA-DR. Earlier these two antigens were identified in HIV-1 preparations analyzed in bulk 1. We prepared MNPs coupled with one of the antibodies against Env HIV protein, VRC01, and captured with these MNPs HIV-1 virions (HIV-1LAI.04 or HIV-1SF162 produced in peripheral blood mononuclear cells (PBMCs)). In addition, we stained these virions with 2G12, another anti-Env antibody. Flow virometry showed a high heterogeneity of HIV-1 virions regarding the presence of LFA-1 and HLA-DR. On average 16.7 &#177;2.0% (n = 6) and 8.6 &#177;0.3% (n = 3) of HIV-1LAI.04 virions carried LFA-1 and HLA-DR, respectively. Only 4.8 &#177;0.6% (n = 3) of all virions were positive for both antigens. On the other HIV-1 strain, HIV-1SF162, these antigens were present on 20.0 &#177;4.4% (n = 6) and 10.8 &#177;1.3% (n = 6) of virions, respectively, whereas both antigens were carried by 6.5 &#177;0.4% (Figure 1).
The distribution of the cellular proteins was dependent on the cells that replicated virus. HIV-infected Jurkat cells produced virions antigenically different from those produced by infected PBMCs. Virions of HIV-1LAI 0.4 produced by Jurkat cells carried 1.6 &#177;1.4% (n = 3) and 1.6&#177;0.7% (n = 4) LFA-1 and HLA-DR respectively. For HIV-1SF162 virions produced in Jurkat cells, these parameters were 7.2 &#177;2.5% (n = 3) and 5.6 &#177;2.7% of (n = 4). Thus, antigenic makeup (at least for HLA-DR and LFA-1) was different for HIV-1LAI.04 produced by two different cell types (p &#60;0.02).
Flow virometry as applied to evaluation of Dengue virus
DENV maturation is associated with the expression of prM protein on the virions. We applied flow virometry to evaluate what fraction of DENV produced in BHK-1 and in LoVo cells constitutes mature viruses. Virions were stained with a lipidic dye DiI. Analysis of individual captured virions revealed prM on 48.2 &#177;5.3% (n = 8) of DENVs. In contrast, 84.5 &#177;3.4% (n = 4) of DENV produced in LoVo cells carried prM. The rest of the virions, respectively 51.8 &#177;5.3% (n = 8) and 15.5 &#177;3.4% (n = 4) were prM negative (Figure 2). Thus, flow virometry can be used to distinguish fully mature individual DENV from immature (or partially mature) DENV virions.
Extracellular vesicles released into bloodstream of healthy volunteers and patients with acute coronary syndrome (ACS)
In order to investigate different EV subsets in patients with ACS and healthy controls, we captured EVs from platelet poor plasma (PPP) by fluorescent MNPs-coupled with antibodies against CD31, CD41a, or CD63. EVs captured by CD31-MNPs were stained for CD41a and CD63, EVs captured by CD41a-MNPs were stained for CD31 and CD63, and EVs captured by CD63-MNPs were stained for CD31 and CD41a (Figure 3).
The amount of EVs captured by CD31-MNPs and positive for one or two of the detection antibodies in ACS patients was 3,359 [2,328; 5,472] EVs/&#181;l in comparison with 1,272 [714; 2,157] EVs/&#181;l in healthy volunteers (p = 0.001). The total amount of EVs captured by CD63 in ACS patients in comparison with healthy volunteers was 3,541 [1,318; 5,173] EVs/&#181;l vs. 806 [488; 2,112] EVs/&#181;l (p = 0.007). There were 4,752 [3,238; 7,173] EVs/&#181;l in plasma of patients with ACS captured by CD41a-MNPs, whereas in plasma of healthy volunteers this number was significantly lower, 2,623 [1,927; 4,188] EVs/&#181;l (p = 0.015). In general, our results indicate that, while EV amounts were mostly increased in ACS patients, the magnitude of the increase was different in different sub-populations of EVs.
<img alt="Figure 1" src="/files/ftp_upload/55020/55020fig1.jpg" /> 
Figure 1: Selective incorporation of cellular antigens in HIV-1 virions replicating in different cell types. HIV (HIV-1LAI.04 or HIV-1SF162) virions released by PBMCs or Jurkat cells were captured with VRC01-MNPs and stained with second anti-gp120 antibody 2G12 and with specific antibodies against HLA-DR and LFA-1. The stained preparations were subjected to flow analysis for HLA-DR (white bars) and LFA-1 (black bars). Means &#177; SEM of three to six experiments 9. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55020/55020fig2.jpg" /> 
Figure 2: Maturation state of DENV virions. DENV produced in BHK-21 cells (A, B) or in LoVo cells (C, D) were labeled with the lipidic dye DiI and, after ultracentrifugation in density gradient medium, stained for prM protein with 2H2 antibodies (A, C) or with isotype control IgG2a (B, D). The labeled virions were then captured with 3H5-1-MNPs and subjected to flow analysis 14. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55020/55020fig3.jpg" /> 
Figure 3: Analysis of EVs from ACS patients and healthy controls. Plasma EVs were captured with CD31-MNPs, CD63-MNPs or CD41a-MNPs and stained with antibodies against CD41a and CD63, against CD31 and CD41a and against CD31 and CD63, respectively. Captured EVs, stained with at least one of the detection Abs, were visualized and enumerated in flow analysis. Data presented as dot plot with median and interquartile range (IQR). Green symbols: healthy volunteers (controls), brown symbols: ACS patients. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55020/55020fig4.jpg" /> 
Figure 4: Evaluation of the fraction of flow events that represent individual virions. (A) HIV-1. Suspension of virions was divided into two parts and stained either with DiD (left panel) or DiO (middle panel). After mixing, the suspension, that contained two differentially stained virions, was captured with VRC01-MNPs and subjected to flow virometry (right panel). Aggregates (dual-colored events) constituted less than 10% of total events. (B-D) DENV. Suspension of DiI-stained virions was serially diluted two fold from 1:2 to 1:256 and acquired with HTS on a flow cytometer (B). DiI-DENVs were labeled with 2H2 antibodies (C) or DiI-DENVs were captured with 3H5-1-MNPs (D). Preparations C and D were then serially diluted two fold from 1:2 to 1:256 and acquired with HTS 14. Virions do not seem to aggregate since in all cases there is a linear correlation between the dilution factor and the number of the events. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55020/55020fig5.jpg" /> 
Figure 5: Detection of single particles by flow cytometer. DiI-stained DENV suspension was serially diluted two fold from 1:2 to 1:2,048 and acquired using an HTS on a flow cytometer. (A) Number of viruses detected as a function of the dilution factor. (B) Median fluorescence intensity (MFI) of DiI labeled DENV 14. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55020/55020fig6.jpg" /> 
Figure 6: Evaluation of the fraction of virions and extracellular vesicles captured with MNPs coupled to specific antibodies. Labeled HIV-1 virions or EVs were captured with MNPs coupled to specific antibodies and subjected to flow virometry. After separation on magnetic columns, the flow-through (not retained) fractions were analyzed for the presence of virions or EVs of interest that were missed in the first run. (A) Labeled HIV-1 BaL virions were captured with 2G12-MNPs (left panel). The flow-through fraction was recaptured and subjected to flow virometry (right panel). In the first cycle about 95% of the virions of interest were captured. (B) Labeled EVs were captured with anti-CD81 MNPs and isolated on magnetic columns (left panel). The flow-through fraction was re-captured and analyzed (right panel). In the first cycle about 99% of the vesicles of interest were captured 8. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55020/55020fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles at JoVE.com

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

flow-virometry-to-analyze-antigenic-spectra-virions-extracellular

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Video: Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from one cell to another. EVs have been shown to travel short and far distances in the body and are tissue-specific. EVs are not only found in tissues, but they can also be found in practically all bodily fluids, such as tears, saliva, cerebral spinal fluid, blood, etc. Even though EVs can be collected non-invasively from tears and saliva, only small volumes can be collected at a time, which can cause issues in obtaining enough EVs to analyze proteins. The scanner discussed in this paper is a nanoparticle analyzer that provides a solution to&#160;this problem, allowing us to characterize and study the phenotype, size, and total particle count of EVs from as little as 1 &#181;L of biological fluid. This protocol will expand the knowledge of EVs from small volumes of samples that are difficult to extract from patients. This could enhance patient comfort and potentially identify new therapeutic targets for a range of diseases and disorders.

Here, we demonstrate the characterization method for extracellular vesicles (EVs) collected from biological fluids, such as tears and saliva, of human subjects. The scanner used in this method is capable of detecting the phenotype, size, and total particle count of EVs from 1 &#181;L of the sample.

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Summary

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Chapters in this video

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Characterizing Extracellular Vesicles from Biological Fluids