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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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><tbody><tr><td>IMDM</td><td>Gibco</td><td>12440061</td><td></td></tr><tr><td>Amicon Ultra-15, PLHK Ultracel-PL Membran, 100&amp;nbsp;kDa&amp;nbsp;</td><td>Millipore</td><td>UFC910024</td><td></td></tr><tr><td>CytoFlex, Flow Cytometer Platform</td><td>Beckman Coulter</td><td>CytoFlex</td><td></td></tr><tr><td>DMEM, high glucose, HEPES, no phenol red</td><td>Gibco</td><td>21063045</td><td></td></tr><tr><td>Dulbecco&#039;s PBS (PBS) Ca- and Mg-free</td><td>Lonza</td><td>BE17-512F</td><td></td></tr><tr><td>ExoCap CD63 Capture Kit</td><td>JSR Life Sciences</td><td>Ex-C63-SP</td><td></td></tr><tr><td>ExoCap CD81 Capture Kit</td><td>JSR Life Sciences</td><td>Ex-C81-SP</td><td></td></tr><tr><td>ExoCap CD9 Capture Kit</td><td>JSR Life Sciences</td><td>Ex-C9-SP</td><td></td></tr><tr><td>Exosome-Depleted FBS</td><td>Thermofisher</td><td>A2720801</td><td></td></tr><tr><td>Exosome-depleted FBS Media Supplement</td><td>SBI</td><td>EXO-FBS-250A-1</td><td></td></tr><tr><td>FBS-Fetal Bovine Serum</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>FITC anti-human CD9 Antibody</td><td>Biolegend</td><td>312104&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; RRID: AB_2075894</td><td></td></tr><tr><td>Flow Cytometer analysis software</td><td>Beckman Coulter</td><td>Kaluza</td><td></td></tr><tr><td>NanoSight LM10</td><td>Malvern</td><td>NanoSight LM10</td><td></td></tr><tr><td>NanoSight Software</td><td>Malvern</td><td>NTA 2.3</td><td></td></tr><tr><td>Optima Max-XP</td><td>Beckman Coulter</td><td>393315</td><td></td></tr><tr><td>PE anti-human CD63 Antibody</td><td>Biolegend</td><td>353004&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; RRID:AB_10897809</td><td></td></tr><tr><td>PE anti-human CD81 (TAPA-1) Antibody</td><td>Biolegend</td><td>349505&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; RRID:AB_10642024</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>5382 000 015</td><td></td></tr><tr><td>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 ...

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

Universidad de Cartagena UDEC

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

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Biochemistry

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Neuroscience

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

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 ...

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method&#39;s limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

This protocol describes a method for the isolation of urinary extracellular vesicles, uEVs, from healthy human donors and their phenotypic characterization by the size and surface marker expression using flow cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

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

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

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

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 ...

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

Setting a Successful Sorting for Extracellular Vesicle Isolation

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications.
First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. 
In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique.
Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

This protocol provides a detailed description of sorting extracellular vesicles (EVs) released by mesenchymal stromal cells. In particular, it focuses on the instrument setting and the optimization of the sorting conditions. The goal is to sort extracellular vesicles while preserving their characteristics.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. ...

Characterizing Extracellular Vesicles from Biological Fluids

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

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

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