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 collecte...

<ol>
	<li>Barile, L., Vassalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Therapy+delivery+tools+and+biomarkers+of+diseases.">Exosomes: Therapy delivery tools and biomarkers of diseases.</a> Pharmacology &amp; Therapeutics. , (2017).</li><li>Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+secreted+vesicles+and+intercellular+communications.">Exosomes: secreted vesicles and intercellular communications.</a> Molecular Biology Reports. 3, 15 (2011).</li><li>Yadav, D. 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=Liquid+biopsy+in+pancreatic+cancer:+the+beginning+of+a+new+era.">Liquid biopsy in pancreatic cancer: the beginning of a new era.</a> Oncotarget. 9, 26900-26933 (2018).</li><li>Balbi, 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=First+Characterization+of+Human+Amniotic+Fluid+Stem+Cell+Extracellular+Vesicles+as+a+Powerful+Paracrine+Tool+Endowed+with+Regenerative+Potential.">First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.</a> Stem Cells Translational Medicine. 6, 1340-1355 (2017).</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, (2018).</li><li>Lai, R. 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=Exosome+secreted+by+MSC+reduces+myocardial+ischemia/reperfusion+injury.">Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.</a> Stem Cell Research. 4, (2009).</li><li>Doeppner, T. 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J., Nolte-'t Hoen, E. N., Stoorvogel, W., Arkesteijn, G. J., Wauben, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nature Protocols. 7, 1311-1326 (2012).</li><li>Pospichalova, V., 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=Simplified+protocol+for+flow+cytometry+analysis+of+fluorescently+labeled+exosomes+and+microvesicles+using+dedicated+flow+cytometer.">Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer.</a> Journal of Extracellular Vesicles. 4, 25530 (2015).</li><li>van der Pol, 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=Particle+size+distribution+of+exosomes+and+microvesicles+determined+by+transmission+electron+microscopy,+flow+cytometry,+nanoparticle+tracking+analysis,+and+resistive+pulse+sensing.">Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.</a> Journal of Thrombosis and Haemostasis. 12, 1182-1192 (2014).</li><li>Witwer, K. 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. 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=Low-density+lipoprotein+mimics+blood+plasma-derived+exosomes+and+microvesicles+during+isolation+and+detection.">Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.</a> Scientific Reports. 6, 24316 (2016).</li><li>Sluijter, J. P. 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=Extracellular+vesicles+in+diagnostics+and+therapy+of+the+ischaemic+heart:+Position+Paper+from+the+Working+Group+on+Cellular+Biology+of+the+Heart+of+the+European+Society+of+Cardiology.">Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position Paper from the Working Group on Cellular Biology of the Heart of the European Society of Cardiology.</a> Cardiovascular Research. 114, 19-34 (2018).</li></ol>

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 ...

Extracellular Vesicles are increasingly being used as a diagnostic biomarker and potential therapeutic tool in many field. Flow Cytometric is the most straight forward analytic method for characterizing by suit faced markers this protocol can be used in analyzing an assized particle, is fast, and is applicable to conventional seismometers without the need for special setting or the dedicated instrument. Although this protocol is used to categorize Extracellular Vesicles from said condition and medium, it can be expanded towards categorization of blood arrived AVs, to replace invasive tissue biopsies in patients.For researchers new to the protocol, it is critical to perform steps described in the set-up method section, and to closely follow the demonstrated getting strategies Begin by coating 55 centimeter squared Petri-dishes with 02%porcine skin gelatin, in PBS. After 15 minutes, wash the dish and plate 4.4 times 10 to the fifth cardiac progenitor cells onto each dish in 7 milliliters of Iscove's Modified Dulbecco's Medium, or IMDM, supplemented with 20%fetal bovine serum, and 1%penicillin and streptomycin for their incubation at 37 degrees Celsius and 5%carbon dioxide. When the cells reach about 80%confluency, wash the cultures two times with Dulbecco's PBS without calcium and magnesium and feed the cells with 10 milileters of serum free exosome production medium.After 7 days, pool the conditioned medium from each plate, into a single polypropylene centrifuge tube and centrifuge the medium to remove any cell debris. Transfer the supernatent into a 100 kilodalton centrifuge filter tube, And concentrate the cleared, conditioned, medium with an additional centrifugation. Transfer the concentrate into a micro centrifuge tube for a final benchtop centrifugation and add the supernatent into a polycarbonate thick wall micro centrifuge tube.Fill the tube with PBS, to a final volume of 3.2 milileters, and load the sample into a titanium fixed angeled rotor. Then load the rotor into a tabletop ultracentrifuge for ultracentrifugation, and resuspend the pellet in 100 microliters of fresh PBS. For nanoparticle tracking and analysis quantification, dilute 1 microliter of the ultracentrifuged sample, in 999 microliters of PBS, and load the sample into a 1 milileter syringe without bubbles.Load the syringe into the inlet port of the examination chamber, and turn on the laser. Use the capture button to open the camera, and adjust the focus. Then record at least three different frames of 60 seconds each, and use the batch process option in the software, to analyze the three different acquisitions.To prepare the sample for acquisition, first add the appropriate experimental monoclonal antibody conjugated capture beads at a 1:1:1 ratio, into a micro centrifuge tube, and vortex the resulting capture bead pool. Next, add the appropriate volume of sample to 1 microliter of capture bead pool to generate a 1 times 10 to the 8 particles plus 1.2 times 10 to the 5th beads per test concentration. Add 1 microliter of beads to a tube labeled beads as the negative control, and adjust the volume in each tube to 100 microliters with fresh PBS.Then place the tubes in a thermo mixer, with shaking, at 400 rotations per minute, and 4 to 10 Celsius, overnight. The next day, transfer the samples to a round bottom 96 well plate, and add the appropriate florescence conjugated antibodies to each well. After a 1 hour incubation at 4 to 10 degrees Celsius, add 100 microliters of PBS to each well, and open the acquisition software.Select cytometer and system start up program, to start the instrument and open a new experiment. Create an experimental template, and select plate and add plate. Select the position of the samples on the plate, and click set as sample well.Enter the sample name in the naming rules, open the channel tab, and select the appropriate florescence signal channels. Now load the plate into the instrument. And click dot plot, to create a new forward scatter area versus side scatter area dot plot.Select initialize to start the laser and the fluidics and click run to start the acquisition. Adjust the scale to show the population in the middle of the forward versus side scatter plot, and draw a beads gate around the whole sample population to exclude the debris. Click stop and dot plot to create a forward scatter height versus forward scatter area dot plot, and gate the sample on the beads population.Draw a new gate around the bigger population and name it Singlets, and click dot plot to create one fluorescent signal versus side scatter area dot plot, per experimental fluorophore used. Gate the new dot plot on the Singlets. Then select the number of events to record, and select record to start the experimental acquisition.At the end of the analysis, load the acquisition files into the appropriate analysis software, and open a new protocol. Create dot plots for each file as just demonstrated, and set all of the scatter axis to linear scale, and all of the florescent axis to log scale. Then show the florescence geometric mean in the relevant signals channels, to calculate the full change in mean florescence intensity, in the different extracellular vesicle preparations.After testing different conditions to set the smallest total amount of extracellular vesicles to reach the early exponential phase of a mean florescence intensity curve, the minimum number of particles was determined to be 1 times 10 to the 8 particles per staining. The optimal concentration of antibody for 1 times 10 to the 8 particles to achieve the highest signal without nonspecific antibody binding, was determined to be 10 micrograms per milliliter for both anti CD9_FITC and anti CD63_PE antibodies and five micrograms per milliliter for the anti CD81_PE antibody. To confirm the suitability of the method, for analyzing cup shaped extracellular vesicles, and not membrane debris, the extracellular vesicles were sonicated, resulting in a decrease in mean florescence intensity, after as little as 30 seconds of sonication, with no florescence detected at all after one minute of mechanical disruption.X's own purification from cardiac progenitor cells as demonstrated, yields extracellular vesicles, expressing low levels of CD9 and high levels of CD63 and CD81 from both cell populations. Furthermore, while X's own surface markers are clearly identifiable without ultracentrifugation, this enrichment step greatly enhances the mean florescence intensity of these markers. When working with extracellular vesicles, there's more careless nano particle pellets can be easily missed.Therefore, it is essential to follow all the step as demonstrated in the protocol. Using extracellular vesicles as a biomarker is now one of the most emerging field research. And soon, may be translated into the clinical diagnostic used as an alternative screening tool.

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13.4K visualizações.  Cardiocentro Ticino Foundation, Switzerland. Vesículas extracelulares estão sendo cada vez mais utilizadas como biomarcadores diagnósticos e potencial ferramenta terapêutica em muitos campos. Flow Cytometric é o método analítico mais direto para caracterizar por marcadores de terno este protocolo pode ser usado na análise de uma partícula assized, é rápido e é aplicável a sismômetros convencionais sem a necessidade de configuração especial ou o instrumento dedicado. Embora este protocolo seja usado para categorizar vesí...