Name: purification of high yield extracellular vesicle preparations away from virus
Uploaded: 2019-09-12T14:00:00
Description: Watch this Scientific Journal Video about Purification of High Yield Extracellular Vesicle Preparations Away from Virus at JoVE.com

We would like to thank all members of the Kashanchi lab, especially Gwen Cox. This work was supported by National Institutes of Health (NIH) Grants (AI078859, AI074410, AI127351-01, AI043894, and NS099029 to F.K.).

1. Filtration and Precipitation of Extracellular Vesicles (EVs)
<ol>
	<li>To prepare the culture supernatant from infected or transfected cells (i.e., cell lines and/or primary cells), culture approximately 10 mL of late-log cells for 5 days at 37 &#176;C and 5% CO2 in appropriate culture medium (i.e., RPMI or DMEM with 10% fetal bovine serum [FBS]). 
	NOTE: All culture medium reagents should be free of EVs, and can be either purchased (see Table of Materials) or prepar...

<ol>
	<li>Taylor, D. D., Shah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolating+extracellular+vesicles+impact+down-stream+analyses+of+their+cargoes.">Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.</a> Methods (San Diego, Calif). 87, 3-10 (2015).</li><li>Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Extracellular+Vesicles:+General+Methodologies+and+Latest+Trends.">Isolation of Extracellular Vesicles: General Methodologies and Latest Trends.</a> BioMed Research International. 2018, 8545347 (2018).</li><li>Momen-Heravi, F., 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=Current+methods+for+the+isolation+of+extracellular+vesicles.">Current methods for the isolation of extracellular vesicles.</a> Biological Chemistry. 394 (10), 1253-1262 (2013).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Current Protocols in Cell Biology. , (2006).</li><li>Boriachek, K., 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=Biological+Functions+and+Current+Advances+in+Isolation+and+Detection+Strategies+for+Exosome+Nanovesicles.">Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles.</a> Small (Weinheim an Der Bergstrasse, Germany). 14 (6), (2018).</li><li>DeMarino, C., 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=Antiretroviral+Drugs+Alter+the+Content+of+Extracellular+Vesicles+from+HIV-1-Infected+Cells.">Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells.</a> Scientific Reports. 8 (1), 7653 (2018).</li><li>Van Deun, J., 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=The+impact+of+disparate+isolation+methods+for+extracellular+vesicles+on+downstream+RNA+profiling.">The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.</a> Journal of Extracellular Vesicles. 3, (2014).</li><li>Lobb, R. 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A., 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=Exosomes+from+uninfected+cells+activate+transcription+of+latent+HIV-1.">Exosomes from uninfected cells activate transcription of latent HIV-1.</a> The Journal of Biological Chemistry. 292 (28), 11682-11701 (2017).</li><li>Pleet, M. 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=Ebola+VP40+in+Exosomes+Can+Cause+Immune+Cell+Dysfunction.">Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.</a> Frontiers in Microbiology. 7, 1765 (2016).</li><li>Pleet, M. 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=Ebola+Virus+VP40+Modulates+Cell+Cycle+and+Biogenesis+of+Extracellular+Vesicles.">Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles.</a> The Journal of Infectious Diseases. , (2018).</li><li>Anderson, M. 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=Viral+antigens+detectable+in+CSF+exosomes+from+patients+with+retrovirus+associated+neurologic+disease:+functional+role+of+exosomes.">Viral antigens detectable in CSF exosomes from patients with retrovirus associated neurologic disease: functional role of exosomes.</a> Clinical and Translational Medicine. 7 (1), 24 (2018).</li><li>Vlassov, A. V., Magdaleno, S., Setterquist, R., Conrad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+current+knowledge+of+their+composition,+biological+functions,+and+diagnostic+and+therapeutic+potentials.">Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.</a> Biochimica Et Biophysica Acta. 1820 (7), 940-948 (2012).</li><li>Th&#233;ry, C., Zitvogel, L., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+composition,+biogenesis+and+function.">Exosomes: composition, biogenesis and function.</a> Nature Reviews. Immunology. 2 (8), 569-579 (2002).</li><li>Schwab, A., 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=Extracellular+vesicles+from+infected+cells:+potential+for+direct+pathogenesis.">Extracellular vesicles from infected cells: potential for direct pathogenesis.</a> Frontiers in Microbiology. 6, 1132 (2015).</li><li>Narayanan, A., 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=Exosomes+derived+from+HIV-1-infected+cells+contain+trans-activation+response+element+RNA.">Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.</a> The Journal of Biological Chemistry. 288 (27), 20014-20033 (2013).</li><li>Sami Saribas, A., Cicalese, S., Ahooyi, T. M., Khalili, K., Amini, S., Sariyer, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Nef+is+released+in+extracellular+vesicles+derived+from+astrocytes:+evidence+for+Nef-mediated+neurotoxicity.">HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity.</a> Cell Death &amp; Disease. 8 (1), e2542 (2017).</li><li>Yang, 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=Exosomal+miR-9+Released+from+HIV+Tat+Stimulated+Astrocytes+Mediates+Microglial+Migration.">Exosomal miR-9 Released from HIV Tat Stimulated Astrocytes Mediates Microglial Migration.</a> Journal of Neuroimmune Pharmacology: The Official Journal of the Society on NeuroImmune Pharmacology. 13 (3), 330-344 (2018).</li><li>Arakelyan, A., Fitzgerald, W., Zicari, S., Vanpouille, 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=Extracellular+Vesicles+Carry+HIV+Env+and+Facilitate+Hiv+Infection+of+Human+Lymphoid+Tissue.">Extracellular Vesicles Carry HIV Env and Facilitate Hiv Infection of Human Lymphoid Tissue.</a> Scientific Reports. 7 (1), 1695 (2017).</li><li>Jaworski, E., 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=Human+T-lymphotropic+virus+type+1-infected+cells+secrete+exosomes+that+contain+Tax+protein.">Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.</a> The Journal of Biological Chemistry. 289 (32), 22284-22305 (2014).</li><li>Heinemann, M. 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=Benchtop+isolation+and+characterization+of+functional+exosomes+by+sequential+filtration.">Benchtop isolation and characterization of functional exosomes by sequential filtration.</a> Journal of Chromatography. A. 1371, 125-135 (2014).</li><li>McNamara, R. 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=Large-scale,+cross-flow+based+isolation+of+highly+pure+and+endocytosis-competent+extracellular+vesicles.">Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles.</a> Journal of Extracellular Vesicles. 7 (1), 1541396 (2018).</li><li>Heinemann, M. L., Vykoukal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+Filtration:+A+Gentle+Method+for+the+Isolation+of+Functional+Extracellular+Vesicles.">Sequential Filtration: A Gentle Method for the Isolation of Functional Extracellular Vesicles.</a> Methods in Molecular Biology. 1660, 33-41 (2017).</li><li>Busatto, 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=Tangential+Flow+Filtration+for+Highly+Efficient+Concentration+of+Extracellular+Vesicles+from+Large+Volumes+of+Fluid.">Tangential Flow Filtration for Highly Efficient Concentration of Extracellular Vesicles from Large Volumes of Fluid.</a> Cells. 7 (12), (2018).</li><li>Andriolo, G., 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=Exosomes+From+Human+Cardiac+Progenitor+Cells+for+Therapeutic+Applications:+Development+of+a+GMP-Grade+Manufacturing+Method.">Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method.</a> Frontiers in Physiology. 9, 1169 (2018).</li><li>Pleet, M. 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The outlined method allows for enhanced EV yield and the separation of virus from EVs using a combination approach to isolation. Relatively large quantities of starting material (i.e., cell supernatant) can be filtered prior to EV isolation by precipitation, DG separation, and nanoparticle enrichment, resulting in a final volume of ~30 &#181;L, allowing for immediate usage in a variety of downstream assays. The use of nanoparticle enrichment is essential as, compared to traditional ultracentrifugation, these EV-enriching...

Research centered around extracellular vesicles (EVs), specifically exosomes, a type of EV ranging 30-120 nm and characterized by the presence of three tetraspanin markers CD81, CD9, and CD63, has largely been shaped by the development of methods to isolate and purify the vesicles of interest. The ability to dissect multifaceted mechanisms has been hindered due to complex and time-consuming techniques which generate samples composed of a heterogeneous population of vesicles generated via different pathways with a wide range of contents, sizes, and densities. While this is an issue for nearly all EV research, it is of particular importance when studying EVs in the context of viral infection, as virions and virus-like particles (VLPs) can be similar in diameter to the vesicles of interest. For example, the Human Immunodeficiency Virus Type 1 (HIV-1) is approximately 100 nm in diameter, which is roughly the same size as many types of EVs. For this reason, we have designed a novel EV isolation workflow to address these issues.
The current gold standard of EV isolation is ultracentrifugation. This technique makes use of the various vesicle densities, which allows the vesicles to be separated by centrifugation with differential sedimentation of higher density particles versus lower density particles at each stage 1,2. Several low-speed centrifugation steps are required to remove intact cells (300-400 x g for 10 min), cell debris (~2,000 x g for 10 min), and apoptotic bodies/large vesicles (~10,000 x g for 10 min). These initial purifications are followed by high speed ultracentrifugation (100,000-200,000 x g for 1.5-2 h) to sediment EVs. Wash steps are performed to further ensure EV purity, however, this results in the reduction of the number of isolated EVs, thereby lowering total yield 3,4. This method&#8217;s utility is further limited by the requirement of a large number of cells (approximately 1 x 108) and a large sample volume (&#62; 100 mL) to achieve adequate results.
To address the growing concerns, precipitation of vesicles with hydrophilic polymers has become a useful technique in recent years. Polyethylene glycol (PEG), or other related precipitation reagents, allows the user to pull down the vesicles, viruses, and protein or protein-RNA aggregates within a sample by simply incubating the sample with the reagent of choice, followed by a single low-speed centrifugation1,2,5. We have previously reported that use of PEG or related methods to precipitate EVs in comparison to traditional ultracentrifugation results in a significantly higher yield6. This strategy is fast, easy, does not require additional expensive equipment, is readily scalable, and retains EV structure. However, due to the promiscuous nature of this method, the resulting samples contain a variety of products including free proteins, protein complexes, a range of EVs, and virions thus requiring further purification to obtain the desired population1,2,7,8.
To overcome the heterogeneity of EVs obtained from various precipitation methods, density gradient ultracentrifugation (DG) is utilized to better separate particles based upon their density. This method is carried out using a stepwise gradient using a density gradient medium, such as iodixanol or sucrose, which allows for the separation of EVs from proteins, protein complexes, and virus or virus-like particles (VLPs). It is important to note that, while it was once thought that DG allowed for more precise separation of EV subpopulations, it is now known that sizes and densities of various vesicles can overlap. For example, exosomes are known to have flotation densities of 1.08-1.22 g/mL9, while vesicles isolated from the Golgi (COPI+ or clathrin+) have densities of 1.05-1.12 g/mL and those from the endoplasmic reticulum (COPII+) sediment at 1.18-1.25 g/mL1,2,3,4,9. Additionally, if one desires to compare exosomal fractions against fractions containing viral particles, this may become more difficult depending upon the density of the virus of interest&#8212;there are viruses other than HIV-1 that likely equilibrate at the same densities as exosomal positive fractions2.
Finally, enrichment of EV preps for downstream visualization and functional assays is vital to EV research. The use of EV-enriching nanoparticles, specifically, multi-functional hydrogel particles that range 700-800 nm in diameter, are a critical step in achieving concentrated EV preps. They possess a high affinity aromatic bait which encapsulated by a porous outer sieving shell to promote selectivity. The nanoparticles utilized in this study include two distinct preparations with different core baits (Reactive Red 120 NT80; and Cibacron Blue F3GA NT82) which have shown to increase capture of EVs from various reagents and biofluids (see the Table of Materials)6,10,11,12,13,14,15. The particles offer easy enrichment of EVs from numerous starting materials including iodixanol fractions, cell culture supernatant, as well as patient biofluids such as plasma, serum, cerebral spinal fluids (CSF), and urine6,13.
The method presented here improves the efficiency of current EV purification techniques by combining several technologies; EV precipitation, density gradient ultracentrifugation, and particle capture, to streamline the workflow, reduce sample requirements, and increase yield to obtain a more homogenous EV sample for use in all EV research. This method is particularly useful in the investigation of EVs and their contents during viral infection as it includes a 0.22 &#181;m filtration step to exclude large, unwanted vesicles and VLPs and separation of the total EV population based on density to effectively isolate EVs from virions.

<table>
	<tbody>
		<tr>
			<td>CEM CD4+ Cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>117</td>
			<td>CEM</td>
		</tr>
		<tr>
			<td>DPBS without Ca and Mg (1X)</td>
			<td>Quality Biological</td>
			<td>114-057-101</td>
			<td></td>
		</tr>
		<tr>
			<td>ExoMAX Opti-Enhancer</td>
			<td>Systems Biosciences</td>
			<td>EXOMAX24A-1</td>
			<td>PEG precipitation reagent</td>
		</tr>
		<tr>
			<td>Exosome-Depleted FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Peak Serum</td>
			<td>PS-FB3</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>HIV-1 infected U937 Cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>165</td>
			<td>U1</td>
		</tr>
		<tr>
			<td>Nalgene Syringe Filter 0.2 &#181;m SFCA</td>
			<td>Thermo Scientific</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanotrap (NT80)</td>
			<td>Ceres Nanosciences</td>
			<td>CN1030</td>
			<td>Reactive Red 120 core</td>
		</tr>
		<tr>
			<td>Nanotrap (NT82)</td>
			<td>Ceres Nanosciences</td>
			<td>CN2010</td>
			<td>Cibacron Blue F3GA core</td>
		</tr>
		<tr>
			<td>Optima XE-980 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D1556-250mL</td>
			<td>Iodixanol</td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Tube, 14x89mm</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of high yield extracellular vesicle preparations away from virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

PEG precipitation increases EV yield 
Our combination approach to EV isolation is significantly more efficient in terms of EV recovery as compared to traditional ultracentrifugation, as evident by the 90% reduction in the volume of starting material required. Ultracentrifugation, the current gold standard in EV isolation, requires approximately 100 mL of culture supernatant to produce an adequate EV prep for downstream assays, whereas our novel protocol requires only...

Watch this Scientific Journal Video about Purification of High Yield Extracellular Vesicle Preparations Away from Virus at JoVE.com

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

This protocol is significant because it concentrates extracellular vesicles, EVs, through a combination of technologies, while allowing for separation of virions away from EVs. This method maximizes EV recovery above the current gold standard of ultracentrifugation for multiple downstream analysis and characterization of virus-free EV preps. This protocol is ideal for the study of EV's and viral infections.This method can be adapted to other virus systems, such as HTLV, Ebola, Zika, and more. I would expect a first time user of this method to struggle with nanoparticle preparation, so we recommend carefully following our specifications. Some optimization may be necessary, depending on the system.Visual demonstration of this method is important for illustrating the overall work flow and nuances of the protocol, which will reduce time and mistakes for first time users. Start by preparing culture supernatant from infected or transfected cells. Culture approximately 10 milliliters of late log cells for five days at 37 degrees Celsius and 5%carbon dioxide, making sure that all medium reagents are free of extracellular vesicles.When the culture is ready, pellet the cells by centrifugation at 3, 000 times G for five minutes and discard the pellet. Filter the supernatant with a sterile 0.22 micrometer filter and collect the filtrate in a clean tube. Add an equal volume of PEG precipitation reagents to the filtrate, and invert the tube several times to mix.Incubate the mixture at four degrees Celsius overnight. After the incubation, centrifuge the mixture at 1500 times G for 30 minutes, to yield a heterogeneous extracellular vesicle, or EV, pellet. Discard the supernatant, and re-suspend the pellet in 150 to 300 microliters of 1X PBS, without calcium and magnesium.Keep the pellet on ice, while preparing a density gradient. Prepare the iodixanol density gradient medium with 11 density fractions, ranging from six to 18%iodixanol, as described in the manuscript. Mix each tube by vortexing and layer the density fractions into a clean and dry swinging bucket ultracentrifuge tube.Add the re-suspended EV pellet to the top of the layered gradient and ultracentrifuge the tube at 10, 000 times G, and four degrees Celsius for 90 minutes. Carefully transfer each fraction from the ultracentrifuge tube to a new microcentrifuge tube. Prepare a 30%slurry of nanoparticles for EV fraction enrichment, by mixing equal volumes of NT80, NT82 and 1X-PBS.Vortex the nanoparticle mixture to ensure homogeneity. Add 30 microliters of the slurry to each density fraction and mix by either pipetting or inverting the tubes. The most critical step is the addition of the nano-articles to concentrate EV's, following the density gradient separation.Without this, EV recovery is poor. Rotate the nanoparticle containing density fractions overnight, at four degrees Celsius. Then centrifuge the density fractions at 20, 000 times G for five minutes at room temperature.Discard the liquid and wash the EV pellet twice with 1X-PBS. To prepare the pellet for RNA isolation, re-suspend it 50 microliters of autoclaved, deionized water, filtered and treated with depsy, according to the manuscript directions. The RNA can then be isolated, using a commercial kit.For analysis with gel electrophoresis, re-suspend the pellet in 15 microliters of Laemmli buffer. Heat the sample to 95 degrees Celsius for three minutes. Repeat the heating step two more times, vortexing gently and spinning the sample down between heat cycles.Centrifuge the sample for 15 seconds at 20, 000 times G, and load the supernatant directly onto the gel. For best results, limit the amount of particles loaded onto the gel, and run it at 100 volts, to prevent any loaded nanoparticles from entering the gel. PEG precipitation allows for significantly more efficient EV recovery, than traditional ultracentrifugation.When using 10 milliliters of culture, this approach results in an approximately 500 fold higher yield than ultracentrifugation. This approach also results in an increased isolation efficiency of exosomes, which is evident through higher levels of exosome marker proteins. Western PLA analysis shows, a 3000 fold increase in CD81, a four fold increase in CD63 and a 40 fold increase in CD9.Furthermore, this protocol allows for downstream studies of EV mediated mechanisms in HIV1 infection, by isolating EV's from verions. Western PLA analysis shows that EV's are found in three fraction populations. While the virus is present in only two of them.EV's infraction 10.8 through 12 are free of virus contamination. The most important thing to remember is that the nanoparticles are crucial for optimal EV enrichment. Their addition is a must.Many downstream assays, such as western blot, PCR, pure diomech analysis by mass spectrometry in plate-based cellular assays can be performed. These allow for characterization, mechanistic, and/or functional studies. This technique allows for specific studies that examine EV cargo and functionality without contaminating viral background.This provides a foundation for studying EV's in pathogenesis and development of potential therapeutics. To overcome the limitations of this technique, and to apply EV preparation and isolation to large volumes, we recommend the use of advanced systems, such as tangential flow filtration. The most hazardous instrument to use is the ultracentrifuge, during the density gradient separation.Make sure that all centrifuge tubes weight the same, to avoid imbalance and potential rotor failure.

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...