L.B. was supported by research grants of Helmut Horten Stiftung and Velux Stiftung, Zurich (Switzerland). G.V. was supported by research grants of Swiss National Science Foundation, the Cecilia-Augusta Foundation, Lugano, and the SHK Stiftung f&#252;r Herz- und Kreislaufkrankheiten (Switzerland)

1. Collection and Processing of Conditioned Media
<ol>
	<li>Coat 55 cm2 Petri dishes with 0.02% porcine skin gelatin in PBS.</li>
	<li>Plate CPC (8,000/cm2) in pre-coated dishes with 7 mL of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) supplemented with 20% FBS (Fetal Bovine Serum) and 1% Penicillin/Streptomycin (P/S). 
	NOTE: The term &#8220;CPC&#8221; refers to human explant derived cells that have been described elsewhere14. CM can be collected from different cell types cultured in specific culture conditions. Wear gloves and work under a biological hood.</li>
	<li>Once cells reach about 80% confluence, remove the culture medium, wash cells twice with Dulbecco&#39;s PBS (PBS) Ca- and Mg-free and replace it with 10 mL of serum-free Exo-production medium (Dulbecco&#39;s Modified Eagle&#39;s Medium [DMEM] High Glucose, 4.5 g/mL). 
	NOTE: Proceed with a progressive decrease of serum concentration in culture medium, to gradually adapt cells to serum-free condition (SF). For cells that are sensitive to serum-free medium prepare a medium containing FBS but depleted of EV. Prepare medium supplemented with all nutrients, plus 10%&#8211;20% (v/v) FBS. Centrifuge the medium overnight at 100,000 &#215; g, 4 &#176;C. Be aware that this procedure does not guarantee 100% removal of FBS-derived EV.</li>
	<li>After 7 days, collect the CM in a polypropylene centrifuge tubes. 
	NOTE: The time for medium conditioning depends on the cell type. For cells that are sensitive to serum-free medium, increase the initial volume of medium and decrease the time for CM collection to 24-48 h.</li>
	<li>Clear CM from cell debris by centrifuge at 3,000 x g for 20 min at 4&#8211;10 &#176;C. Collect the supernatant in a 100 kDa centrifuge filter tube.</li>
	<li>Concentrate cleared CM by spinning the tube at 2,000 x g for 20 min at 4&#8211;10 &#176;C. 
	NOTE: This step will reduce the initial volume of CM allowing the use of small-volume tubes for high speed centrifuge steps and will contribute to eliminate small protein aggregates.</li>
	<li>Collect the concentrate in a microcentrifuge tube. Centrifuge at 10,000 x g for 15 min at 4&#8211;10 &#176;C.</li>
	<li>Collect the supernatant and proceed to section 2 (EV enrichment). If an ultracentrifuge is not available proceed directly to section 3 (Nanoparticle Tracking Analysis (NTA) quantification). 
	NOTE: Pre-enrichment of EV fraction improves final read-out in fluorescence intensity (see representative results and Figure 4). Cleared CM (from point 1.8) can be stored at 4 &#176;C for no longer then 1-2 days or alternatively at &#8722;80&#176;C for several months.5 CM should be pre-cleared to remove any cellular debris before storage at -80&#176;C in order to ensure that exosome-sized debris is not formed in the freeze-thaw process.</li>
</ol>
2. Extracellular Vesicles Enrichment by Ultracentrifuge (Optional)
<ol>
	<li>Place the supernatant from step 1.8 in a polycarbonate thick wall ultracentrifuge tube. Fill the tube with 1x phosphate buffer saline (PBS) until it reaches the maximal capacity (3.2 mL).</li>
	<li>Load samples in a titanium fixed-angle rotor (8 x 3.2 mL, k-Factor 13). Load the rotor in a tabletop ultracentrifuge. Ultracentrifuge at 100,000 x g for 3 h at 4&#8211;10 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 100 &#181;L of 1x PBS.</li>
</ol>
3. Nanoparticle Tracking Analysis (NTA) Quantification
<ol>
	<li>Dilute 1 &#181;L of sample (from either step 2.3 or 1.8) in 999 &#181;L of 1x PBS. Load the sample in a 1 mL syringe, avoiding bubble formation. Load the syringe in the inlet port of the examination chamber.</li>
	<li>Turn on the laser. Open the camera with the Capture button. Adjust the focus.</li>
	<li>Record at least 3 different frames of 60 s each.</li>
	<li>Analyze the 3 different acquisitions using the Batch Process option in the software. 
	NOTE: If the NTA technology is not available, perform a protein quantification by the Bradford assay (1 x 108 particles correspond to 1&#8211;2 &#181;g of total protein after ultracentrifugation or to 50&#8211;60 &#181;g total protein if quantification is performed directly for CM as it includes proteins contaminants different from EV; see Table 1)5.</li>
</ol>
4. Sample Preparation
<ol>
	<li>Prepare a pool of the 3 types of capture beads (CD9 beads, CD63 beads, and CD81 beads at a 1:1:1 ratio; see Table of Materials) and vortex. 
	NOTE: The pool has been tested to be stable for 1 month. A single type of capturing bead may be used, this will allow the detection of EV sub-populations not discriminable with pool of beads. By using single antigen capturing beads, it will be possible to contemporary demonstrate the presence of tetraspanin proteins, such as CD9, CD81, or CD63, and the antigen of interest detected by fluorescent antibody. Alternative aldehyde-sulfate latex beads are also commercially available. These beads present hydrophobic surface for the adsorption of MV and EV.</li>
	<li>In a round bottom tube, add the amount of volume corresponding at 1 x 108 particles plus 1 &#181;L of the bead pool, corresponding to 1.2 x 105 beads in total (for each test).</li>
	<li>Prepare a tube containing 1 &#181;L of beads without EV that will serve as negative control, hereby referred as &#8220;Beads&#8221;.</li>
	<li>Adjust the volume to 100 &#181;L with 1x PBS. Place the tube in a thermo-mixer, and shake at 400 rpm overnight at 4&#8211;10 &#176;C.</li>
	<li>The next day, move the samples to a round bottom 96-well plate (or FC tubes).</li>
	<li>Add the antibody (10 &#181;g/mL of CD9_FITC, 10 &#181;g/mL of CD63_PE, and 5 &#181;g/mL of CD81_PE).</li>
	<li>Incubate 1 h at 4&#8211;10 &#176;C.</li>
	<li>Add 100 &#181;L of 1x PBS to each well.</li>
	<li>Proceed to acquisition.</li>
</ol>
5. Acquisition
<ol>
	<li>Open the acquisition software and start up the instrument (Cytometer &#62; System Startup Program). Open a new experiment.</li>
	<li>Create an experimental template, then select Plate and then Add Plate. Select the position of the samples on the plate and then press Set As Sample Well.</li>
	<li>Enter the sample name (i.e. CPC#1_CD9FITC) in the Naming Rules. Open the Channel Tab and switch on the FITC and PE Channels.</li>
	<li>Load the plate in the instrument.</li>
	<li>Press Dot Plot and create a new dot plot (P1). Set Forward Scatter Area (FSC-A) versus Side Scatter-Area (SSC-A).</li>
	<li>Select Initialize to start the laser and the fluidics. Press Acquire to start the acquisition.</li>
	<li>Adjust the scale to show the population in the middle of the P1. In P1 draw the &#8220;BEADS&#8221; gate around the whole population (excluding debris) (Figure 2A). Press Stop.</li>
	<li>Press Dot Plot and create a new dot plot (P2). Set Forward Scatter-Height (FSC-H) versus FSC-A. Gate the new dot plot on &#8220;BEADS.&#8221; In P2 draw a new gate around the bigger population and name it &#8220;SINGLETS&#8221; (Figure 2A). 
	NOTE: This strategy is designed in order to avoid as much as possible the inclusion of beads aggregates in both acquisition and analysis steps.</li>
	<li>Press Dot Plot and create two different dot plots; one FITC versus SSC-A and one PE versus SSC-A. Gate the new dot plot on &#8220;SINGLETS.&#8221;</li>
	<li>Select the number of Events to record (e.g., 20,000). Select Record and start the experiment.</li>
</ol>
6. Data Analysis
<ol>
	<li>Load the acquisition files into the analysis software. 
	NOTE: The following steps have to be applied to every sample including beads only and every unknown sample.</li>
	<li>Open a new protocol and create dot plots following the same strategy described in step 5.</li>
	<li>Set all scatter axes to linear scale and all the fluorescence axes to log scale.</li>
	<li>Show the Fluorescence Geometric Mean (X-Gmean-MFI) in FITC and PE channels.</li>
	<li>Calculate the ratio X-Gmean of the samples / X-Gmean of the beads stained with antibody without exosome.</li>
	<li>Compare the fold change MFI of different EV preparations.</li>
</ol>
7. Extracellular Vesicle Number Titration
NOTE: Sections 7 to 10 can be performed to set up the number of particles, antibodies concentration and specificity, but can be skipped if the cell type, from which EV are derived, and antibodies remain the same.
<ol>
	<li>Dilute particles, obtained from step 2.3, in 1x PBS to obtain a series of suspensions ranging from 5 x 105 to 2.5 x 108 particles.</li>
	<li>For each suspension add in a round bottom tube 1 &#181;L of capture bead pool.</li>
	<li>Follow the protocol from step 4.3.</li>
</ol>
8. Antibody Titration
NOTE: Antibody titration is usually performed by adding a single antibody for each tube.
<ol>
	<li>Keep the number of EV (total particle or protein content) constant in all samples. 
	NOTE: The appropriate number is empirically determined as described above in section 7.</li>
	<li>Follow the protocol from steps 4.1 to 4.5.</li>
	<li>Test a range of antibody concentrations above and below the amount recommended by the supplier. For example, for an antibody with a suggested concentration of 10 &#181;g/mL per test, test 1, 2, 5, 10, 20 and 50 &#181;g/mL. Include a sample with &#8220;beads only&#8221;, adding the highest concentration of antibody.</li>
	<li>Incubate for 1 h at 4&#8211;10 &#176;C.</li>
	<li>Add 100 &#181;L of 1x PBS for each sample.</li>
	<li>Acquire the samples.</li>
</ol>
9. Incubation Time
<ol>
	<li>Verify the incubation time performing acquisitions at different time points (e.g., 1 h, 2 h, and 5 h).</li>
	<li>Follow the protocol starting from section 4.</li>
	<li>Incubate samples at 4&#8211;10 &#176;C for 1 h.</li>
	<li>Acquire samples.</li>
	<li>Incubate samples at 4&#8211;10 &#176;C for 3 h.</li>
	<li>Acquire samples.</li>
</ol>
10. Protocol Validation
<ol>
	<li>EV binding
	<ol>
		<li>In a round bottom tube, add the amount of volume corresponding to 1 x 108 particles or the corresponding amount of total protein.</li>
		<li>Adjust the volume to 100 &#181;L with 1x PBS.</li>
		<li>Disrupt the EV membrane by sonication (alternatively a heat shock step can be applied) according to manufacturer&#8217;s instructions. Set frequency and intensity. The sonication period can be empirically established setting a time-course experiment (i.e., 0 s, 30 s, 1 min, 5 min, etc.).</li>
		<li>Add 1 &#181;L of the bead pool.</li>
		<li>Follow the protocol starting from step 4.4.</li>
	</ol>
	</li>
	<li>Antibody specificity
	<ol>
		<li>Prepare one tube for each fluorochrome-conjugated IgG and add the complex of beads-EV as described in step 4.6. 
		NOTE: The fluorochrome-conjugated Isotype must match with the IgG of the used antibody.</li>
	</ol>
	</li>
</ol>

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W., 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=Updating+the+MISEV+minimal+requirements+for+extracellular+vesicle+studies:+building+bridges+to+reproducibility.">Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility.</a> Journal of Extracellular Vesicles. 6, 1396823 (2017).</li><li>Thery, 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 Cellular Biology. , (2006).</li><li>Suarez, H., 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=A+bead-assisted+flow+cytometry+method+for+the+semi-quantitative+analysis+of+Extracellular+Vesicles.">A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles.</a> Scientific Reports. 7, 11271 (2017).</li><li>Sodar, B. 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Nothing to declare.

Conventional FC technique remains the most straightforward analytic method to characterize markers expressed onto the surface of EV. In this regard, selecting the most appropriate protocol is crucial to obtain useful information on individual particle fractions of interest by avoiding limitations due to instrument sensitivity. We described a method using magnetic particles coupled with antibodies that recognize Exo and small EV surface antigens which are suitable for downstream FC application. We validated the method usi...

Cells secrete extracellular vesicles (EV) of different sizes including microvesicles (MV) and exosomes (Exo). The latter can be distinguished from MV by both size and the subcellular compartment of origin. MV (200&#8211;1,000 nm in size) are released from parent cells by shedding from the plasma membrane. Conversely, Exo (30&#8211;150 nm) originate from endosomal membranes and are released into the extracellular space when the multivesicular bodies (MVB) fuse with the cell membrane1,2.
EV are increasingly used as diagnostic biomarkers as well as, potentially, therapeutic tools in many fields including oncology, neurology, cardiology, and musculoskeletal diseases3,4,5. A vast majority of ongoing studies using EV as therapeutic agents exploit the isolation of vesicles from cell-conditioned medium (CM) of in vitro cultured cells. Mesenchymal stem cells (MSCs) exert beneficial effects in several contexts, and MSC-derived EV have shown benefits in models of myocardial ischemia/reperfusion injury6 and brain injury7. MSC-derived EV also exhibit immune modulatory activities that can be exploited to treat immune rejection, as demonstrated in a model of therapy-refractory graft-versus-host disease8. Amniotic fluid stem cells (hAFS) actively enrich CM with MVs and Exo, heterogeneously distributed in size (50&#8211;1,000 nm), which mediate several biological effects, such as proliferation of differentiated cells, angiogenesis, inhibition of fibrosis, and cardioprotection4. We have recently shown that EV, and particularly Exo, secreted by human cardiac-derived progenitor cells (Exo-CPC) reduce myocardial infarct size in rats5,9.
Exo share a common set of proteins on their surface, including tetraspanins (CD63, CD81, CD9) and major histocompatibility complex class I (MHC-I). In addition to this common set of proteins, Exo also contain proteins specific for the EV subset of the producer cell type. Exo markers are gaining paramount importance because they play a crucial role in inter-cellular communication, thereby regulating many biological processes5,10. Because of their small size, finding an easy way to analyze EV using classical flow cytometry (FC) remains a challenging task.
Here, we present a simplified protocol for EV analysis using FC, which can be applied to pre-enriched samples obtained through ultracentrifugation or directly to CM (Figure 1). The method uses beads coated with a specific antibody that binds canonical Exo-associated surface epitopes (CD63, CD9, CD81) without additional washes. FC analyses can be performed using a conventional cytometer with no need for adjustments prior to measurements. Methods for the characterization of antigens on individual small particles using flow cytometers have been described by other groups with respect to various applications11,12,13. Here, we used functionalized magnetic beads for the capture of small particles and Exo, followed by phenotyping of captured particles by FC. Although this method can be used to characterize the antigenic composition of small vesicles released by any cell type in vitro, here we provided specific cell culture conditions that apply for the culture of human cardiac progenitor cells (CPC) and the most appropriate environment for the production of EV by these cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:816px;" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">IMDM</td>
			<td style="width:113px;">Gibco</td>
			<td style="width:235px;">12440061</td>
			<td style="width:99px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15, PLHK Ultracel-PL Membran, 100&#160;kDa&#160;</td>
			<td>Millipore</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CytoFlex, Flow Cytometer Platform</td>
			<td>Beckman Coulter</td>
			<td>CytoFlex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, HEPES, no phenol red</td>
			<td>Gibco</td>
			<td>21063045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s PBS (PBS) Ca- and Mg-free</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD63 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C63-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD81 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C81-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoCap CD9 Capture Kit</td>
			<td>JSR Life Sciences</td>
			<td>Ex-C9-SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome-Depleted FBS</td>
			<td>Thermofisher</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome-depleted FBS Media Supplement</td>
			<td>SBI</td>
			<td>EXO-FBS-250A-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS-Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-human CD9 Antibody</td>
			<td>Biolegend</td>
			<td>312104&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID: AB_2075894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer analysis software</td>
			<td>Beckman Coulter</td>
			<td>Kaluza</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight LM10</td>
			<td>Malvern</td>
			<td>NanoSight LM10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoSight Software</td>
			<td>Malvern</td>
			<td>NTA 2.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima Max-XP</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE anti-human CD63 Antibody</td>
			<td>Biolegend</td>
			<td>353004&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID:AB_10897809</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE anti-human CD81 (TAPA-1) Antibody</td>
			<td>Biolegend</td>
			<td>349505&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; RRID:AB_10642024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer C</td>
			<td>Eppendorf</td>
			<td>5382 000 015</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">TLA-110</td>
			<td>Beckman Coulter</td>
			<td>TLA-110 rotors</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Total number of particles for single staining
Since a single bead can bind more than one particle, we tested different conditions to set the smallest amount of total EV (single antibody per tube) to reach the early exponential phase of MFI curve. A fixed concentration of antibody was used while the total number of particles ranged from 5 x 105 to 2.5 x 108. As shown in ...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media at JoVE.com

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

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 ...

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13.5K Views.  Cardiocentro Ticino Foundation, Switzerland. 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.

Video: Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

Quantitative Analysis of Microbial Autoaggregation Using Imaging Flow Cytometry

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Author Spotlight: Advancing Research in Microbial Autoaggregation Using Imaging Flow Cytometry

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

A Flow Cytometry Protocol for Understanding Export Mechanisms

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...