Name: Setting a Successful Sorting for Extracellular Vesicle Isolation (Video) | JoVE Journal
Uploaded: 2024-10-11T14:30:00.000+00:00
Duration: 8 min 37 s
Description: Learn to isolate high-purity extracellular vesicles from mesenchymal stromal cells using optimized cell sorter settings. This protocol preserves EV integrity and enables identification of rare populations for tissue regeneration research.

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5 minutes

We thank Emanuele Canonico for technical support. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory established by IRCCS Ospedale San Raffaele and Universit&#224; Vita-Salute San Raffaele. The work of Enrico Ragni and Laura de&#160;Girolamo was supported by the Italian Ministry of Health, &#34;Ricerca Corrente&#34;.

Fluorescence-Based Sorting of Adipose-Derived Extracellular Vesicles

<ol><li>Welsh, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+information+for+studies+of+extracellular+vesicles+%28misev2023%29%3A+From+basic+to+advanced+approaches%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Minimal information for studies of extracellular vesicles (misev2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404(2024).</li>
<li>Xu, R., Greening, D. W., Zhu, H. J., Takahashi, N., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicle+isolation+and+characterization%3A+Toward+clinical+application%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Extracellular vesicle isolation and characterization: Toward clinical application.</a> J Clin Invest. 126 (4), 1152-1162 (2016).</li>
<li>Dixson, A. C., Dawson, T. R., Di Vizio, D., Weaver, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Context-specific+regulation+of+extracellular+vesicle+biogenesis+and+cargo+selection%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Context-specific regulation of extracellular vesicle biogenesis and cargo selection.</a> Nat Rev Mol Cell Biol. 24 (7), 454-476 (2023).</li>
<li>Witwer, K. W., Wolfram, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles+versus+synthetic+nanoparticles+for+drug+delivery%5BTitle%5D)+AND+%22Nat+Rev+Mater%22%5BJournal%5D)">Extracellular vesicles versus synthetic nanoparticles for drug delivery.</a> Nat Rev Mater. 6 (2), 103-106 (2021).</li>
<li>Cheng, L., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutically+harnessing+extracellular+vesicles%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Therapeutically harnessing extracellular vesicles.</a> Nat Rev Drug Discov. 21 (5), 379-399 (2022).</li>
<li>Du, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles%3A+A+rising+star+for+therapeutics+and+drug+delivery%5BTitle%5D)+AND+%22J+Nanobiotechnology%22%5BJournal%5D)">Extracellular vesicles: A rising star for therapeutics and drug delivery.</a> J Nanobiotechnology. 21 (1), 231(2023).</li>
<li>Keshtkar, S., Azarpira, N., Ghahremani, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell-derived+extracellular+vesicles%3A+Novel+frontiers+in+regenerative+medicine%5BTitle%5D)+AND+%22Stem+Cell+Res+Ther%22%5BJournal%5D)">Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine.</a> Stem Cell Res Ther. 9 (1), 63(2018).</li>
<li>Orbay, H., Tobita, M., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+isolated+from+adipose+and+other+tissues%3A+Basic+biological+properties+and+clinical+applications%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Mesenchymal stem cells isolated from adipose and other tissues: Basic biological properties and clinical applications.</a> Stem Cells Int. 2012, 461718(2012).</li>
<li>Caplan, A. I., Dennis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+as+trophic+mediators%5BTitle%5D)+AND+%22J+Cell+Biochem%22%5BJournal%5D)">Mesenchymal stem cells as trophic mediators.</a> J Cell Biochem. 98 (5), 1076-1084 (2006).</li>
<li>Rani, S., Ryan, A. E., Griffin, M. D., Ritter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell-derived+extracellular+vesicles%3A+Toward+cell-free+therapeutic+applications%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Mesenchymal stem cell-derived extracellular vesicles: Toward cell-free therapeutic applications.</a> Mol Ther. 23 (5), 812-823 (2015).</li>
<li>Galipeau, J., Sensébé, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stromal+cells%3A+Clinical+challenges+and+therapeutic+opportunities%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Mesenchymal stromal cells: Clinical challenges and therapeutic opportunities.</a> Cell Stem Cell. 22 (6), 824-833 (2018).</li>
<li>Jia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+extracellular+vesicles+isolation+and+separation%3A+Current+techniques%2C+pending+questions+and+clinical+applications%5BTitle%5D)+AND+%22Theranostics%22%5BJournal%5D)">Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications.</a> Theranostics. 12 (15), 6548-6575 (2022).</li>
<li>Ohnuma, K., Yomo, T., Asashima, M., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sorting+of+cells+of+the+same+size%2C+shape%2C+and+cell+cycle+stage+for+a+single+cell+level+assay+without+staining%5BTitle%5D)+AND+%22BMC+Cell+Biol%22%5BJournal%5D)">Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining.</a> BMC Cell Biol. 7, 25(2006).</li>
<li>Morales-Kastresana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-fidelity+detection+and+sorting+of+nanoscale+vesicles+in+viral+disease+and+cancer%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.</a> J Extracell Vesicles. 8 (1), 1597603(2019).</li>
<li>Mortati, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+study+of+extracellular+vesicles+migration+in+cartilage-derived+osteoarthritis+samples+using+real-time+quantitative+multimodal+nonlinear+optics+imaging%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">In vitro study of extracellular vesicles migration in cartilage-derived osteoarthritis samples using real-time quantitative multimodal nonlinear optics imaging.</a> Pharmaceutics. 12 (8), 734(2020).</li>
<li>Andreu, Z., Yanez-Mo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tetraspanins+in+extracellular+vesicle+formation+and+function%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Tetraspanins in extracellular vesicle formation and function.</a> Front Immunol. 5, 442(2014).</li>
<li>Mildmay-White, A., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+surface+markers+on+adipose-derived+stem+cells%3A+A+systematic+review%5BTitle%5D)+AND+%22Curr+Stem+Cell+Res+Ther%22%5BJournal%5D)">Cell surface markers on adipose-derived stem cells: A systematic review.</a> Curr Stem Cell Res Ther. 12 (6), 484-492 (2017).</li>
<li>Welsh, J. A., Tang, V. A., Van Der Pol, E., Gorgens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MIFlowCyt-EV%3A+The+next+chapter+in+the+reporting+and+reliability+of+single+extracellular+vesicle+flow+cytometry+experiments%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">MIFlowCyt-EV: The next chapter in the reporting and reliability of single extracellular vesicle flow cytometry experiments.</a> Cytometry A. 99 (4), 365-368 (2021).</li>
<li>Maia, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Employing+flow+cytometry+to+extracellular+vesicles+sample+microvolume+analysis+and+quality+control%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Employing flow cytometry to extracellular vesicles sample microvolume analysis and quality control.</a> Front Cell Dev Biol. 8, 593750(2020).</li>
</ol>

The authors have nothing to disclose.

Analyzing and sorting EVs is challenging due to their small size and the fact that they are near the detection limit of most flow cytometers. Our objective was to develop a protocol for isolating EVs derived from AMSCs labeled with CFSE. CFSE was selected as the staining method due to its reported high EVs labeling efficiency (&#8805;90%), without the formation of unwanted particles such as protein aggregates given by antibodies. Nevertheless, it is possible that few EVs without esterase could be missed and future studies will be necessary to identify staining methods able to label these particles for sorting protocols19.
We aimed to establish sorting conditions that allow for the collection of pure EVs without altering their physical properties. This was achieved by leveraging the technical capabilities of the sorter used and implementing specific measures, such as reducing the standard sorting pressure according to the nozzle size. We established and validated these sorting conditions by using FSC polystyrene beads. Subsequently, we applied this protocol to ASC EVs, repeating the experiment four times and consistently achieving similar results, thereby confirming the repeatability of our protocol.
However, it is important to note some critical aspects and limitations, which we outline here. (i) Purity of the sorted sample: it is challenging to conclusively prove the purity of the sorted CFSE-positive EVs, as the background noise of the instrument always appears during re-analysis. However, we can assume that we have enriched the CFSE-positive population. The absence of CFSE-positive events in the CFSE-negative population indicates that our sorting process was successful, indirectly demonstrating the purity of the sorted sample. (ii) Potential contamination of the sorted sample: we cannot rule out the possibility that the sorted fraction may contain small vesicles that are undetectable by the instrument and may be inadvertently included in the sorting drops. (iii) Integrity of EVs post-sorting: We are aware that the pressure applied by the instrument could potentially damage the vesicles. To mitigate this, we used a 70 &#181;m nozzle with a lower pressure of 35 psi instead of the standard 60 psi. Additionally, we carefully prepared the samples, avoiding ultracentrifugation steps that could alter the physical characteristics of EVs. Despite these precautions, we cannot guarantee that the sorted EVs remain intact and unaltered. Therefore, we recommend including post-sorting analysis and, when possible, functionality tests to assess the condition of the EVs.
Here are some possible modifications and troubleshooting to the protocol. This protocol has been set by using an instrument that allows the change of pressures; however, for instruments with stricter settings, it is recommended to use a 100 &#181;m nozzle to have a lower pressure in order to preserve the integrity of the EVs as much as possible.
Despite these considerations, we assert that our protocol offers significant advantages compared to other methods for EV separation. Firstly, it allows for the isolation of specific populations of EVs. While we chose to stain the vesicles with CFSE - a fluorescent dye that identifies structures with intact cell membranes - it is also possible to sort specific EV populations using monoclonal antibodies. In such cases, additional staining controls are essential. The International Society for Extracellular Vesicles (ISEV), the International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) provide comprehensive guidelines for conducting reproducible flow cytometry experiments. Detailed instructions can be found in the MISEV 20231 and MIFlowCyt -EV guidelines18. Secondly, this procedure is less time-consuming than other methods that involve multiple ultracentrifugation steps.
In our opinion, this protocol could be a successful strategy for separating and collecting a specific population of EVs that could potentially be useful for therapeutic purposes.

Extracellular Vesicles (EVs) are a heterogeneous group of membrane-structured vesicles released by almost all cells, delimited by a lipid bilayer, unable to replicate on their own1. They can be found in several biofluids such as blood plasma, serum, saliva, breast milk, urine, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites2. One of the main functions of EVs is to transport various molecules, including nucleic acids, proteins, lipids, and carbohydrates, between a donor and a recipient cell. This can occur through various mechanisms, such as direct membrane fusion, receptor-ligand interaction, endocytosis, and phagocytosis3,4. For this reason, they have been demonstrated to play an important role in a lot of physiological and pathological processes, and they show considerable promise as novel biomarkers of disease, as drug delivery vehicles, and as therapeutic agents5,6.
Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from many tissues, including adipose tissue, dental pulp, umbilical cord blood, placenta, amniotic fluid, Wharton&#39;s jelly, and even the brain, lung, thymus, pancreas, spleen, liver, and kidney. In recent years, they have attracted considerable interest in regenerative medicine7. Adipose-derived mesenchymal stem cells (ASCs) can be harvested from fat tissue through a less invasive procedure compared to other sources like bone marrow, resulting in lower risks of severe complications and avoiding ethical issues8.
Additionally, adipose tissue contains a significantly higher concentration of MSCs than bone marrow (1% versus &#62;0.01%) and other sources such as the dermis, dental pulp, umbilical cord, and placenta. MSCs are crucial in the regeneration of injured tissues and cells due to their differentiation ability and their secretion of a broad repertoire of growth factors, chemokines, and cytokines; these therapeutic benefits are attributable to their differentiation ability but also to the fact that they secrete a broad repertoire of growth factors, chemokines, and cytokines. A striking example is given by MSCs&#39; therapeutic potential for orthopedic conditions, with the term &#34;Musculoskeletal Diseases&#34; having the higher number of registered clinical studies under clinicaltrials.gov (accessed 13th May 2024).
Moreover, MSCs can also secrete EVs that take part in tissue regeneration via transferring information to damaged cells or tissue and exert biological activity similar to the mother cells9,10. For this reason, MSC-EVs may be a valuable substitute for cell therapy to achieve a cell-free approach11, with two clinical studies involving MSC-EVs for orthopedic conditions (NCT05261360 and NCT04998058). However, several challenges still exist for the clinical applications of EVs. For example, there are some concerns about EV isolation techniques: most of them do not guarantee vesicle purity or integrity. Moreover, some isolation techniques are complex, time-consuming, and have low repeatability, making them unsuitable for clinical use12.
Cell sorting, on the other hand, is a commonly used method that allows for the isolation of single cells from heterogeneous cell suspensions by using specific fluorescent markers13. It can be used for many applications and adapted to different sample types. However, although cell sorting is a well-established and widely used technology, EV sorting is still very challenging because most EVs are below the minimum detection threshold for even the most sensitive flow cytometers. There are some features that make a sorter more suitable for this purpose. First of all, using a Jet-in-air system in which the stream suspending the particles is interrogated by lasers in air, rather than within a flow cell; this system preserves the sample by decreasing the stress to which it is subjected. A second important point is the presence of an &#34;obscuration&#34; bar between the stream and the collection lens that decrease the background optical noise of the instrument. Although it is low, the background noise is not completely eliminated and constitutes a reference that provides a partial window into the events that fall under the threshold: it is very important for the analysis of events that are close to the &#34;limit of detection&#34; of the instrument14. Finally, the sorter features a dual-path Forward Scatter (FSC) with two different masks that allow for improved discrimination between small and large particles in the sample.
Based on this, we developed a protocol aimed to separate carboxyfluorescein succinimidyl ester (CFSE) labeled MSC-EVs by using a high-sensitivity cell sorter. To minimize the manipulation of EVs and preserve their integrity and quantity, we avoided ultracentrifugation steps during the sample preparation. Furthermore, sorting conditions were adjusted to minimize stress on the vesicles, including further optimization of our instrument by reducing the sorting pressure associated with the nozzle size (70 &#956;m nozzle for a pressure of 35 psi).

<table><tbody><tr><td>5(6)-Carboxyfluorescein diacetate&amp;nbsp;N-succinimidyl ester</td><td>Merck</td><td>150347-59-4</td><td></td></tr><tr><td>Adipose Mesenchymal Stromal Cells</td><td>Wepredic, Parc d&#039;affaires, 35760 Saint-Gr&amp;eacute;goire, France</td><td></td><td>Cells used in this study</td></tr><tr><td>Alexa 488 anti-Caveolin</td><td>R&amp;amp;D Systems</td><td>IC5736G</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD44</td><td>BioLegend</td><td>338805</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD63</td><td>BioLegend</td><td>353007</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD81 (TAPA-1)</td><td>BioLegend</td><td>349509</td><td>Flow cytometry antibody</td></tr><tr><td>APC anti-human CD9</td><td>BioLegend</td><td>312107</td><td>Flow cytometry antibody</td></tr><tr><td>BC CytoFLEX S</td><td>Beckman Coulter</td><td></td><td>BC CytoFLEX S equipped with 3 lasers, Blue, Red and Violet</td></tr><tr><td>Flow-Check Pro Fluorospheres</td><td>Beckman Coulter</td><td>A63493</td><td>Fluorescent control beads for MoFLO Astrios EQ</td></tr><tr><td>FlowJo software (version 10.8.1)</td><td>BD</td><td>version 10.8.1</td><td>Analysis software</td></tr><tr><td>IntraSure kit</td><td>BD Biosciences</td><td>641776</td><td>Fixation and permeabilization for intracellular staining</td></tr><tr><td>Megamix-Plus FSC</td><td>BioCytex</td><td>7802</td><td>FSC polystyrene beads&amp;nbsp;</td></tr><tr><td>MoFLO Astrios EQ</td><td>Beckman Coulter</td><td></td><td>MoFLO Astrios EQ equipped with 4 lasers, Blue, Yellow - Green, Violet and Red</td></tr><tr><td>Mouse anti-FLOT1 antibody</td><td>BD Transduction Laboratories</td><td>610820</td><td>Western Blot antibody</td></tr><tr><td>NanoSight NS300</td><td>Malvern</td><td>NS300</td><td></td></tr><tr><td>Rabbit anti-Calnexin antibody</td><td>Origene</td><td>TA336279</td><td>Western Blot antibody</td></tr><tr><td>Rabbit anti-CD9 and CD81 antibody (ExoAb antibody kit)</td><td>System Biosciences</td><td>EXOAB-KIT-1</td><td>Western Blot antibodies</td></tr><tr><td>Rabbit anti-TSG101 antibody&amp;nbsp;</td><td>Merck</td><td>HPA006161</td><td>Western Blot antibody</td></tr><tr><td>Triton X-100</td><td>Merck</td><td>9036-19-5</td><td></td></tr><tr><td>Ultra Rainbow Fluorescent Particles</td><td>Spherotech</td><td>URFP-30-2</td><td></td></tr><tr><td>Ultracel 100 kDa MWCO</td><td>Merck</td><td>UFC910024</td><td></td></tr><tr><td>VER01 - Verity Shells</td><td>Exometry</td><td></td><td>Organo silica beads for scatter calibration</td></tr></tbody></table>

setting a successful sorting for extracellular vesicle isolation

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.

The FSC polystyrene beads have been sorted to validate the instrument setup and sorting conditions. FSC polystyrene beads are a mix of fluorescent beads ranging from 100 nm, 300 nm, 500 nm, and 900 nm and are visible on the FITC channel. Figure 7A shows the SSC log scale versus the FITC log scale dot plot with the four populations of beads before sorting. The fluorescent populations of 100 nm, 300 nm, and 500 nm were gated and sorted. Sorted beads were analyzed for purity and enrichment as an initial proof of concept (Figure 7B). All populations were pure after sorting. The reference background noise is always visible in the dot plots before and after sorting.
In order to meet the requirements listed in MISEV2018/2023 and to demonstrate the nature of vesicles and their degree of purity, experiments were conducted to analyze markers present or absent on EVs. EVs are positive for some specific surface markers15: CD9, CD63, CD81 and CD44. CD9, CD63, and CD81 belong to the tetraspanin family of membrane proteins and are the most common markers to identify exosomes16. CD44 is a marker highly expressed by MSCs and ASCs17.
EVs are also positive for the cytosolic protein Caveolin, as demonstrated by the Intracellular flow cytometry experiment. Additionally, the western blot confirms the positivity for CD9, CD81, TSG101, Flotillin and the negativity for Calnexin (Figure 2).
Next, CFSE labeled ASC-EVs were sorted as reported in the protocol. This sorting procedure was repeated four times, maintaining exactly the same conditions for sample preparation, staining, and sorting (Table 1). In this way, the protocol's repeatability has been demonstrated.
A set of experiments was performed in order to analyze the CFSE-sorted ASC-EVs population and to demonstrate that their characteristics have not changed by sorting. In particular, expression of CD9, CD63, CD81, and CD44 does not change after sorting (Figure 6A).
Another critical point concerns the dimensions of the EVs and how they can change after the sorting procedure due to the stress induced by the pressure. The post-sorting analysis was difficult to perform due to the small amount of sample, but nanoparticle tracking analysis demonstrates that the distribution of the two ASC-EVs samples (pre and post sorting) was the same (Figure 6B). Finally, the analysis of EVs through TEM demonstrates that their morphology seems not significantly altered following sorting (Figure 6C).
Of note, as suggested by MIFlowCyt-EV framework guidelines18, a reduction in the amount of CFSE-positive EVs upon 0.1% Triton X-100 sample treatment was observed, thus supporting their vesicular origin (Figure 8).
<img alt="Flow cytometry dot plots of Megamix sorting; SSC vs FITC; pre-sorting and 100-500 nm size fractions." class="xfigimg" src="/files/ftp_upload/67232/67232fig07.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
Figure 7: FSC polystyrene beads sorting. (A) SSC/FITC dot plot of beads before sorting. SSC/FITC dot plot of re-analyzed beads post sorting: (B) 100 nm sorted beads, (C) 300 nm sorted beads, and (D) 500 nm sorted beads. <a href="https://www.jove.com/files/ftp_upload/67232/67232fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow cytometry results, untreated vs. Triton X-100 treated, CFSE vs. SSC scatter plot analysis." class="xfigimg" src="/files/ftp_upload/67232/67232fig08.jpg" data-altupdatedat="1788304762000" data-alt="Figure 8"> 
Figure 8: EV membrane degradation in the presence of Triton X-100. Triton X-100 at the concentration of 0.1% has been added to the sample for 15 min at room temperature. Representative scatter plots from three independent experiments of untreated (left) and treated (right) samples are reported. <a href="https://www.jove.com/files/ftp_upload/67232/67232fig08large.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>Experiment</td>
			<td>Number of sorted EVs </td>
			<td>Number of CFSE+ sorted EVs</td>
			<td>Pressure (psi)</td>
			<td>Nozzle mm</td>
			<td>Frequency (Hz)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>74 x 106</td>
			<td>4.8 x 106</td>
			<td>35</td>
			<td>70</td>
			<td>66000</td>
		</tr>
		<tr>
			<td>2</td>
			<td>44 x 106</td>
			<td>7 x 106</td>
			<td>35</td>
			<td>70</td>
			<td>66000</td>
		</tr>
		<tr>
			<td>3</td>
			<td>38 x 106</td>
			<td>3 x 106</td>
			<td>35</td>
			<td>70</td>
			<td>66000</td>
		</tr>
		<tr>
			<td>4</td>
			<td>16 x 106</td>
			<td>2 x 106</td>
			<td>35</td>
			<td>70</td>
			<td>66000</td>
		</tr>
	</tbody>
</table>
Table 1: Repeatability of the sorting procedure

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. ...

setting-a-successful-sorting-for-extracellular-vesicle-isolation

Research

JoVE Journal

Biology

Source: DeMarino, C., et al. Purification of High Yield Extracellular Vesicle Preparations Away from Virus. J. Vis. Exp. (2019)The video demonstrates the separation of EVs from virions and other contaminants by density gradient centrifugation, followed by capture and concentration with hydrogel nanoparticles. Washed fractions are then ready for downstream identification of those enriched in EVs.

Separation and Enrichment of Extracellular Vesicles in the Presence of Virions

This video describes the technique of extracting extracellular vesicles from fresh or frozen tissues. The isolated extracellular vesicles can be used for further analysis to uncover their significant roles in disease pathogenesis.

Extracellular Vesicle Extraction: A Technique to Isolate Extracellular Vesicles from Whole Tissue Specimens Using Differential Centrifugation

This video demonstrates the density gradient ultracentrifugation-based purification method to isolate subpopulations of extracellular vesicles suitable for characterization studies.

Purification of Subpopulations of Extracellular Vesicles: A Technique to Purify Subpopulations of Extracellular Vesicles Using Density Gradient Ultracentrifugation

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

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

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

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

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

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.

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

This protocol describes the isolation of small extracellular vesicles from murine skeletal muscle and bone marrow using enzymatic digestion and size-exclusion chromatography, enabling reproducible recovery of vesicle-enriched fractions from structurally complex, low-yield tissues for downstream molecular analysis.

Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Learn to isolate high-purity extracellular vesicles from mesenchymal stromal cells using optimized cell sorter settings. This protocol preserves EV integrity and enables identification of rare populations for tissue regeneration research.

Setting a Successful Sorting for Extracellular Vesicle Isolation

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