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 using two different cell types: primary human CPC that are emerging as a major cell source for Exo-based therapeutic approaches for heart disease; and HEK293 cells, an immortalized cell line widely used in cell biology research because of reliable cell growth and plasticity.
The method can be applied to ultracentrifuge-enriched EV and, for faster analysis, also directly on in vitro cell-derived CM with no pre-enrichment by ultracentrifugation. The starting material is critical when comparing samples. Adding capturing beads directly to the CM will speed up the procedure but at the same time decrease fluorescence intensity, as shown in Figure 4A. It is also critical to use an appropriate amount of PBS to mix beads and EV during the "capturing" step 4.4. When using a constant incubation time, an increased volume will decrease fluorescence intensity due to inefficient EV coupling. 
A limitation of the protocol is that a single capture bead can bind multiple EV/Exo particles on its surface. This will limit the possibility of identifying subsets of EV expressing peculiar combination of antigens using a multiple staining. The bead-based method therefore yields semi-quantitative data. Using beads carrying a single capturing Ab (CD9, CD63 or CD81) will allow at maximum the characterization of particles that express two epitopes: the one present on the bead and recognized by the capturing antibody and the one detected by the antibody that is subsequently added. 
The current gold standard for Exo analyses using FC is a protocol developed by van der Vlist et al. in 201215. It allows for a high-resolution analysis of EV using an optimized configuration of the commercially available high-end FC (e.g., BD Influx). This protocol is extremely detailed and useful but still needs a complex hardware setting with specific FC calibration before use. Three years later, Pospichalova et al.16 proposed a simplified protocol for FC analysis of Exo using a dedicated cytometer specifically developed for analysis of small particles (e.g., Apogee A50 Micro)17. With respect to this protocol and others that have used special threshold setting11, here we propose a basic protocol to perform small EV phenotyping using magnetic binding beads that is suitable for conventional FC instruments and does not require any special setting. Different protocols have described bead-based methods to characterize small EV found in bodily fluids by FC12. Here, we show the immunocapture of discrete sub-populations of vesicles positive for CD9, CD63, and CD81 that are commonly used as Exo markers18. Aldehyde-sulfate latex19,20 and polystyrene12beads remain valid alternatives for binding of EV present in CM and blood plasma fluid; however, aldehyde groups exposed on the surface of the polymer particle enable coupling of unspecific proteins and other materials to the latex particles, thus increasing the risk of contamination by lipoprotein or apoptotic bodies during isolation and detection process21,22. 
Beads used in the protocol bind only entire, well-shaped EV. We proposed to disrupt EV structure by sonication to quench the signal (section 4, "protocol validation"). Indeed, one minute of sonication results in diminished fluorescence intensity, thus showing that a positive signal cannot be affected by membrane debris adsorbed on beads.

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 Figure 3A, the number of particles that allows us to ensure that the antibody performs within an acceptable MFI, avoiding the use of an excess of EV, is 1 x 108 particles/staining.
Antibody titration
We selected the proper concentration of antibody resulting in the highest signal preventing the nonspecific antibody binding. This test has been optimized for 1 x 108 particles, as determined in the previous setting. Anti-CD9_FITC, anti-CD63_PE, and anti-CD81_PE were tested with concentrations ranging from 1 to 50 &#181;g/mL (Figure 3B). Anti-CD9_FITC and anti-CD63_PE antibodies gave a good resolution of signal (7.5 and 130-fold change of MFI vs. beads alone, respectively) when used at concentration of 10 &#181;g/mL while the selected concentration for the anti-CD81_PE antibody was 5 &#181;g/mL (465.3 Fold Change MFI).
Method validation
In order to confirm that our method is suitable to analyze only &#34;cup-shaped&#34; extracellular vesicles and not membrane debris, we applied different sonication steps, at 10% of amplitude, to the solution containing particles. 100 &#181;L of 1x PBS solution containing 1 x 108 particles underwent different sonication steps from 30 seconds to 5 minutes and the resulting preparation containing both broken and well-shaped EV were analyzed as described (experimental protocol from point 3.3). As a result, we found that 30 seconds of sonication decrease MFI that was completely dumped after 1 minute. At this timepoint, no fluorescence is detectable for any of the EV markers (Figure 3D).
Flow cytometry characterization of HEK293- and CPC-derived exosomes
These results were generated following the protocol presented above with the ultracentrifuge isolation method. The isolated exosomes are quantified by NTA technology and loaded overnight with 1 &#181;L of mixed beads (1x anti-CD9:1x anti-CD63:1x anti-CD81). The complex beads + Exo were stained with the proper amount of antibody anti-CD9_FITC, anti-CD63_PE, and anti-CD81_PE (Figure 3E,F and Table 2).
Furthermore, by using EV-CPC we compared FC analysis with or without pre-enrichment by ultracentrifugation. Figure 4 shows that both methods are suitable to profile Exo surface markers. Pre-enrichment of extracellular vesicles fraction greatly improves fluorescence intensity especially for CD63_PE and CD81_PE staining (Figure 4 and Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59128/59128fig1.jpg" /> 
Figure 1: Protocol and NTA plots. (A)&#160;Schematic representation of the experimental protocol. (B) Representative NTA plots for Conditioned Media, Pre-Ultracentrifuge and Post-Ultracentrifuge step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59128/59128fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59128/59128fig2.jpg" /> 
Figure 2: Acquisition and data analysis. (A)&#160;Flow cytometry analysis begins with creating a first gate to the whole beads population &#34;beads&#34; (excluding debris) and then a second gate to distinguish &#34;singlets&#34; events. Singlets are gated on a plot set up with FSC-H as x-axis and FSC-A as y-axis. (B-D) Representative dot plots showing right-shift of fluorescence intensity for the positive populations of beads-exosomes complexes (green, CD9+; red CD63+; brown CD81+). Isotype control (violet) overlap negative gray colored beads. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59128/59128fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59128/59128fig3.jpg" /> 
Figure 3: Titration curves for number of particles (arrows show the selected amount of particles, 1 x 108). (A) Antibody concentrations (arrows show the selected concentration for each used antibody). (B) Both number of particles and antibody concentration are plot vs. mean fluorescence intensity (MFI). (C) Curve showing 3 different acquisition of the preparation with 1&#8211;2 h or 5 h of antibody incubation. (D) Curve showing the decrease of fluorescence following sonication at different time-points. (E-F) Fold change (mean &#177; SD) of MFI for CD9, CD63 and CD81 vs negative control (beads + antibodies, no EV) are shown for HEK293 EV (n = 3 independent replicates) and for CPC EV (n = 3 primary cell lines from 3 different patients) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59128/59128fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59128/59128fig4.jpg" /> 
Figure 4: MFI analysis and comparison between two procedures: direct EV-binding with beads (Capture Beads Isolation) and pre-enrichment with ultracentrifuge (Ultracentrifuge Isolation). Data are shown as fold change (mean &#177; SD) of MFI for (A) CD9, (B) CD63, and (C) CD81 vs. negative control (beads + antibodies, no EV). N = 3 primary cell lines from 3 different patients. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59128/59128fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CPC</td>
			<td>CONC NTA (part/&#181;L)</td>
			<td>CONC (&#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>CPC#1 Pre-Ultracentrifuge</td>
			<td>5.02E+06</td>
			<td>2.01</td>
		</tr>
		<tr>
			<td>CPC#1 Post-Ultracentrifuge</td>
			<td>6.10E+07</td>
			<td>1.04</td>
		</tr>
		<tr>
			<td>CPC#2 Pre-Ultracentrifuge</td>
			<td>5.74E+06</td>
			<td>2.30</td>
		</tr>
		<tr>
			<td>CPC#2 Post-Ultracentrifuge</td>
			<td>7.43E+07</td>
			<td>0.79</td>
		</tr>
		<tr>
			<td>CPC#3 Pre-Ultracentrifuge</td>
			<td>2.02E+06</td>
			<td>1.90</td>
		</tr>
		<tr>
			<td>CPC#3 Post-Ultracentrifuge</td>
			<td>2.91E+07</td>
			<td>0.42</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison between NTA concentration and protein concentration for 3 different patient derived CPC before and after ultracentrifuge.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FOLD CHANGE OF MFI &#177; SD</td>
			<td>CD9</td>
			<td>CD63</td>
			<td>CD81</td>
		</tr>
		<tr>
			<td>HEK293</td>
			<td>24.44 &#177; 19.17</td>
			<td>430.7 &#177; 344.9</td>
			<td>535.2 &#177; 410.3</td>
		</tr>
		<tr>
			<td>CPC#1</td>
			<td>14.15 &#177; 3.72</td>
			<td>236.05 &#177; 43.40</td>
			<td>353.30 &#177; 452.43</td>
		</tr>
		<tr>
			<td>CPC#2</td>
			<td>15.76 &#177; 1.87</td>
			<td>983.06 &#177; 195.63</td>
			<td>374.45 &#177; 108.05</td>
		</tr>
		<tr>
			<td>CPC#3</td>
			<td>8.94 &#177; 7.19</td>
			<td>830.50 &#177; 184.73</td>
			<td>60.05 &#177; 23.18</td>
		</tr>
	</tbody>
</table>
Table 2: Value of fold change (mean &#177; SD) of MFI for HEK293 EV (n = 3 independent replicates) and CPC EV (n = 3 primary cell lines from 3 different patients).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FOLD CHANGE OF MFI &#177; SD</td>
			<td>CD9</td>
			<td>CD63</td>
			<td>CD81</td>
		</tr>
		<tr>
			<td>Capture Beads Isolation</td>
			<td>2.96 &#177; 1.45</td>
			<td>65.65 &#177; 18.87</td>
			<td>21.85 &#177; 6.12</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Isolation</td>
			<td>7.47 &#177; 2.71</td>
			<td>236.00 &#177; 25.06</td>
			<td>65.05 &#177; 13.38</td>
		</tr>
	</tbody>
</table>
Table 3: Value of fold change of MFI (mean &#177; SD) for CPC EV (n = 3 primary cell lines from 3 different patients) isolated by capture beads or ultracentrifuge.
Table 4: Single product specification.

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 ...

flow-cytometric-analysis-extracellular-vesicles-from-cell-conditioned

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K 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

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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 ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

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 ...