Name: Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography (Video) | JoVE Journal
Uploaded: 2026-06-23T12:55:00.000+00:00
Duration: 7 min 26 s
Description: Learn to isolate small extracellular vesicles from mouse skeletal muscle and bone marrow using size-exclusion chromatography. This protocol enables high-purity sEV isolation from minimal tissue input for downstream molecular and functional analyses.

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5 minutes

We thank Jennie Yang, Dariusz &#379;urawek, and Volodymyr Yerko for technical assistance and helpful discussions. We are grateful to the Center for Applied Nanomedicine platform, particularly Nadim Tawil and Laura Montermini, for support with extracellular vesicle characterization and data acquisition. We also thank the animal facility staff, Guylaine Gadoury and Tara Thomson, for their assistance with mouse handling.

<ol>
	<li>Whitham, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+provide+a+means+for+tissue+crosstalk+during+exercise.">Extracellular vesicles provide a means for tissue crosstalk during exercise.</a> Cell Metab. 27 (1), 237-251.e4 (2018).</li><li>Kumar, M. 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I. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+as+a+mediator+of+interorgan+crosstalk+during+exercise:+Implications+for+aging+and+obesity.">Skeletal muscle as a mediator of interorgan crosstalk during exercise: Implications for aging and obesity.</a> Circ Res. 136 (11), 1407-1432 (2025).</li><li>Vanhie, J. J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+exercise-+and+high-fat+diet-induced+bone+marrow+extracellular+vesicles+in+stress+hematopoiesis.">The role of exercise- and high-fat diet-induced bone marrow extracellular vesicles in stress hematopoiesis.</a> Front Physiol. 13, 1054463 (2022).</li><li>Pinto, A. C., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+therapeutic+potential+of+contracting+skeletal+muscle-released+extracellular+vesicles+in+cancer:+Current+insights+and+future+directions.">Exploiting the therapeutic potential of contracting skeletal muscle-released extracellular vesicles in cancer: Current insights and future directions.</a> J Mol Med (Berl). 102 (5), 617-628 (2024).</li><li>Welsh, J. A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+(MISEV2023):+From+basic+to+advanced+approaches.">Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.</a> J Extracell Vesicles. 13 (2), e12404 (2024).</li><li>De Sousa, K. P., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+extracellular+vesicles+and+future+directions+in+diagnosis+and+therapy.">Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy.</a> Wiley Interdiscip Rev Nanomed Nanobiotechnol. 15 (1), e1835 (2023).</li><li>Guo, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+simplified+dichotomic+size-exclusion+chromatography+for+isolating+extracellular+vesicles+toward+clinical+applications.">Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.</a> J Extracell Vesicles. 10 (11), e12145 (2021).</li><li>Ahmadian, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+storage+and+freezing+protocols+for+extracellular+vesicles:+A+systematic+review.">Different storage and freezing protocols for extracellular vesicles: A systematic review.</a> Stem Cell Res Ther. 15 (1), 453 (2024).</li><li>Watanabe, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+releases+extracellular+vesicles+with+distinct+protein+and+microRNA+signatures+that+function+in+the+muscle+microenvironment.">Skeletal muscle releases extracellular vesicles with distinct protein and microRNA signatures that function in the muscle microenvironment.</a> PNAS Nexus. 1 (4), pgac173 (2022).</li><li>Magalhaes-Gama, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+cell-derived+extracellular+vesicles+in+peripheral+blood+and+bone+marrow+of+B-cell+acute+lymphoblastic+leukemia+pediatric+patients:+Proof-of-concept+study.">Exploring cell-derived extracellular vesicles in peripheral blood and bone marrow of B-cell acute lymphoblastic leukemia pediatric patients: Proof-of-concept study.</a> Front Immunol. 15, 1421036 (2024).</li><li>Potter, D. R., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+extracellular+vesicles+attenuate+pulmonary+vascular+permeability+and+lung+injury+induced+by+hemorrhagic+shock+and+trauma.">Mesenchymal stem cell-derived extracellular vesicles attenuate pulmonary vascular permeability and lung injury induced by hemorrhagic shock and trauma.</a> J Trauma Acute Care Surg. 84 (2), 245-256 (2018).</li><li>Osteikoetxea, X., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+detergent+sensitivity+of+extracellular+vesicle+subpopulations.">Differential detergent sensitivity of extracellular vesicle subpopulations.</a> Org Biomol Chem. 13 (38), 9775-9782 (2015).</li><li>Kim, T., Hong, J. W., Lee, L. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+methods+of+isolation+and+purification+of+extracellular+vesicles.">Efficient methods of isolation and purification of extracellular vesicles.</a> Nano Converg. 12 (1), 45 (2025).</li><li>Bracht, J. W. P., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+size-exclusion+chromatography+column+affects+recovery,+purity,+and+miRNA+cargo+analysis+of+extracellular+vesicles+from+human+plasma.">Choice of size-exclusion chromatography column affects recovery, purity, and miRNA cargo analysis of extracellular vesicles from human plasma.</a> Extracell Vesicles Circ Nucl Acids. 5 (3), 497-508 (2024).</li><li>Mo, W., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle-mediated+bidirectional+communication+between+the+liver+and+other+organs:+Mechanistic+exploration+and+prospects+for+clinical+applications.">Extracellular vesicle-mediated bidirectional communication between the liver and other organs: Mechanistic exploration and prospects for clinical applications.</a> J Nanobiotechnology. 23 (1), 190 (2025).</li></ol>

The authors declare no conflicts of interest.

This protocol describes a robust and reproducible approach for isolating sEV-enriched preparations directly from SkM and BM, two low-yield and highly heterogeneous tissues. While EV isolation is well established for biofluids and cell culture systems, standardized methods for tissue-derived EVs remain limited due to challenges associated with tissue dissociation, contamination from intracellular material, and variability in yield. Here, we outline critical steps, troubleshooting strategies, and limitations that influence...

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, lipids, and nucleic acids1. They are released by all cell types and are present in virtually all biological fluids, where they function as key carriers of bioactive cargo, including peptides and micro ribonucleic acids (RNAs)2. Their cargo is selectively sorted, enabling precise delivery of specific signals3, and their vesicular structure allows them to bypass the typical gradient and dilution constraints of freely secreted factors by delivering concentrated molecular payloads over long distances4. Although EVs from biofluids (e.g., plasma, serum, and saliva) provide a convenient snapshot of systemic signaling, they represent a highly mixed population originating from many tissues5. This makes it challenging to determine which organ is producing a specific biological signal or how EV communication is regulated at the tissue level. Because circulating EVs are pooled together, tissue-specific changes can be diluted or masked, limiting the ability to identify the true source of signals6.
Skeletal muscle (SkM) and bone marrow (BM) are major metabolically and functionally active tissues with distinct cellular compositions and signaling roles7,8. Both tissues may release EV populations enriched with unique, tissue-specific cargo; however, these signals can be easily overshadowed in plasma by EVs from high-turnover tissues such as immune cells8,9. Directly isolating EVs from muscle or BM increases sensitivity and enables detection of subtle, tissue-specific adaptations that may not be observable in circulation. Tissue-derived EVs therefore provide essential complementary information, revealing when a specific organ releases EVs, how long those signals persist, and which adaptations are acute versus chronic. Despite increasing interest in EVs, most established isolation methods have been developed for biofluids (e.g., plasma and serum) or cell culture systems. In these contexts, EVs can be obtained with relatively high yield and minimal structural disruption. However, isolating EVs directly from solid tissues such as SkM and BM presents notable technical challenges. These tissues are structurally complex and heterogeneous, requiring mechanical or enzymatic dissociation steps that can introduce artifacts, including high levels of soluble proteins, organelle fragments, and non-vesicular nanoparticles, making it difficult to achieve high-purity EV preparations.
Another major challenge in tissue-derived EV isolation is the lack of standardized workflows for fraction definition and separation10. Differential centrifugation alone often results in co-isolation of protein aggregates and large vesicles, while ultracentrifugation-based methods, although widely used, can lead to vesicle aggregation, deformation, and variable recovery depending on rotor type and experimental conditions. Density gradient approaches improve purity but are time-intensive, low-throughput, and difficult to standardize across laboratories11. Consequently, there is currently no widely adopted, reproducible method optimized for isolating small EVs from solid tissues with high purity and consistent yield10.
Size-exclusion chromatography (SEC) has emerged as a promising alternative for EV isolation because it separates particles based on size without subjecting vesicles to high centrifugal forces. SEC enables efficient separation of small EVs from soluble proteins and low-molecular-weight contaminants while preserving vesicle integrity and biological function. Importantly, SEC allows for clear definition of elution fractions, enabling reproducible identification of EV-enriched fractions and improved consistency across experiments12. However, SEC workflows require careful optimization for tissue-derived samples, particularly with respect to upstream tissue processing, debris removal, and fraction collection. To address these limitations, we developed a reproducible and standardized protocol for isolating sEVs from SkM and BM. This method integrates controlled enzymatic digestion for fibrous tissues, differential centrifugation for debris removal, and SEC-based purification to isolate high-purity vesicles. The workflow is specifically optimized to minimize contamination from intracellular components while preserving vesicle integrity and enabling consistent recovery from low-yield tissues.
This protocol fills a critical methodological gap by providing a practical and scalable approach for isolating tissue-derived sEVs with defined fractionation and validated purity, achieved through nanoparticle tracking analysis (NTA), Western blotting of canonical EV markers, electron microscopy for morphological assessment, and single-particle EV analysis using antibody capture, interferometric sizing, and multichannel fluorescence imaging. It is particularly well suited for studies requiring tissue-specific EV analysis, where biofluid-derived vesicles are insufficient to resolve the cellular origin of signals. Researchers should use this method when the goal is to obtain high-purity sEVs from solid tissues with minimal contamination and preserved vesicle integrity. In contrast, ultracentrifugation-based methods may be preferred for high-yield applications where purity is less critical, and density gradient approaches may be selected when isolating specific EV subpopulations despite lower throughput. Together, this protocol provides a standardized framework for the isolation and characterization of tissue-derived sEVs, enabling reproducible downstream molecular, biochemical, and functional analyses.

<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>Sarstedt</td><td>72.690.300</td><td>General microcentrifuge tube used throughout protocol</td></tr><tr><td>1.5 mL low-protein-binding tube</td><td>Sarstedt</td><td>72.706.600</td><td>Used for EV handling, SEC fraction collection, and storage</td></tr><tr><td>0.2 mL PCR tube</td><td>Fisher Scientific</td><td>14-222-262</td><td>Used for poked-tube bone marrow extraction setup</td></tr><tr><td>16 G needle</td><td>BD</td><td>305197</td><td>Used to puncture the 0.2 mL PCR tube</td></tr><tr><td>30 &amp;micro;m cell strainer</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-098-458</td><td>Used for skeletal muscle sample filtration</td></tr><tr><td>70% ethanol</td><td>Sigma-Aldrich</td><td>1.11727</td><td>Used to sterilize tweezers</td></tr><tr><td>Biosafety cabinet</td><td>NA</td><td>NA</td><td>Used for sterile tissue handling</td></tr><tr><td>Bone cutting instrument</td><td>Fisher Scientific</td><td>10-001-976</td><td>Used to separate femur and tibia and expose marrow</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>BioShop</td><td>ALB001</td><td>Used for Western blot blocking and antibody dilution</td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>BioShop</td><td>CCL302</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Carbon-coated copper grid, 200-mesh</td><td>Sigma-Aldrich</td><td>930342</td><td>Used for TEM sample preparation</td></tr><tr><td>Centrifugal filter, 100 kDa MWCO</td><td>MilliporeSigma</td><td>UFC810096</td><td>Used for EV concentration</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td>Used for skeletal muscle enzymatic digestion</td></tr><tr><td>Cryovial</td><td>Sarstedt</td><td>72.690.300</td><td>Used for tissue and bone storage</td></tr><tr><td>Deionized water (ultrapure)</td><td>NA</td><td>NA</td><td>Used for buffer preparation and TEM washing steps</td></tr><tr><td>DNase I</td><td>Roche</td><td>11284932001</td><td>Used for skeletal muscle enzymatic digestion</td></tr><tr><td>Dry ice</td><td>NA</td><td>NA</td><td>Used for sample cooling and transport</td></tr><tr><td>Enhanced chemiluminescence substrate</td><td>Biorad</td><td>1705061</td><td>Used for Western blot detection</td></tr><tr><td>Filter unit, 0.22 &amp;micro;m</td><td>Sarstedt</td><td>83.3941.501</td><td>Used to filter PBS where applicable</td></tr><tr><td>Forceps / tweezers</td><td>MilliporeSigma</td><td>F3892</td><td>Used for tissue handling</td></tr><tr><td>Glutaraldehyde</td><td>NA</td><td>NA</td><td>Used for TEM fixation</td></tr><tr><td>Glycine</td><td>NA</td><td>NA</td><td>Used in TEM grid washing steps</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14175-095</td><td>Used for DNase I stock preparation</td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.500</td><td>Used for HEPES-NaOH buffer preparation</td></tr><tr><td>Horseradish peroxidase-conjugated streptavidin</td><td>Thermo Fisher</td><td>S911</td><td>Used for Western blot detection</td></tr><tr><td>Imaging system for chemiluminescence</td><td>Biorad</td><td>12003153</td><td>Used to image Western blot membranes</td></tr><tr><td>KCl</td><td>BioShop</td><td>POC308</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>BioShop</td><td>MAG510.500</td><td>Used for HEPES buffer preparation</td></tr><tr><td>2-Methylbutane</td><td>Thermo Fisher</td><td>78-78-4</td><td>Used for skeletal muscle snap-freezing</td></tr><tr><td>Milligram balance</td><td>MilliporeSigma</td><td>Z741359</td><td>Used for tissue and marrow weighing</td></tr><tr><td>NaCl</td><td>BioShop</td><td>SOD004.1</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Nano-particle tracking analysis instrument</td><td>Malvern Panalytical</td><td>NanoSight NS300</td><td>NanoSight NS300 used for particle sizing and counting</td></tr><tr><td>NaOH</td><td>BioShop</td><td>SHY500.500</td><td>Used for pH adjustment</td></tr><tr><td>Nitrocellulose membrane</td><td>BioRad</td><td>1704271</td><td>Used for Western blot transfer</td></tr><tr><td>Nutating mixer</td><td>Labnet</td><td>&amp;nbsp;S0500</td><td>Used during enzymatic digestion</td></tr><tr><td>P1000 pipette</td><td>MilliporeSigma</td><td>FA10006M-1EA</td><td>Used for tissue transfer</td></tr><tr><td>Pipette tip, 1 mL</td><td>Sarstedt</td><td>70.3050.275</td><td>Tip is trimmed to widen opening for tissue fragment transfer</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Wisent</td><td>INC 811.012-FL</td><td>Used throughout protocol; also used for SEC equilibration and elution</td></tr><tr><td>Phosphatase inhibitor</td><td>MilliporeSigma</td><td>4906845001</td><td>Used for Western blot lysate preparation</td></tr><tr><td>Petri dish</td><td>MilliporeSigma</td><td>CLS351146</td><td>Used for bone marrow preparation</td></tr><tr><td>Polyacrylamide gel, 4&amp;ndash;20% gradient stain-free</td><td>BioRad</td><td>4561094</td><td>Used for Western blot separation</td></tr><tr><td>Protease inhibitor</td><td>MilliporeSigma</td><td>11697498001</td><td>Used for Western blot lysate preparation</td></tr><tr><td>RPMI-1640 medium</td><td>Sigma-Aldrich</td><td>R7388-500ML</td><td>Unsupplemented; no FBS or antibiotics</td></tr><tr><td>RIPA buffer, 10&amp;times;</td><td>Merck</td><td>20-188</td><td>Used for protein extraction for Western blotting</td></tr><tr><td>Scalpel, sterile disposable</td><td>Ultident Paragon</td><td>02-90000-10</td><td>Used to mince skeletal muscle tissue</td></tr><tr><td>SEC column, original 70 nm</td><td>IZON</td><td>ICO70P</td><td>Used for skeletal muscle SEC</td></tr><tr><td>SEC column, single 70 nm</td><td>IZON</td><td>ICS70P</td><td>Used for bone marrow SEC</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Added to PBS for washing the column</td></tr><tr><td>Sodium cacodylate buffer</td><td>NA</td><td>NA</td><td>Used for TEM fixation buffer</td></tr><tr><td>Luer Slip Single Use Syringe without Needle&amp;nbsp;</td><td>Medline</td><td>SYR101020</td><td>Used during NTA acquisition if applicable</td></tr><tr><td>Transmission electron microscope</td><td>FEI</td><td>NA</td><td>Tecnai G2 Spirit Twin 120 kV Cryo-TEM used for TEM imaging</td></tr><tr><td>Tween-20</td><td>BioShop</td><td>TWN508.1</td><td>Used in PBS-T for Western blotting</td></tr><tr><td>Uranyl acetate</td><td>NA</td><td>NA</td><td>Used for TEM negative staining</td></tr><tr><td>Whatman filter paper</td><td>MilliporeSigma</td><td>WHA1003055</td><td>Used to wick excess TEM stain</td></tr></tbody></table>

isolation of small extracellular vesicles from murine skeletal muscle and bone marrow by size-exclusion chromatography

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

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, lipids, and ribonucleic acids (RNAs). Isolation of sEVs from solid tissues such as skeletal muscle (SkM) and bone marrow (BM) remains challenging due to low yield and contamination from non-vesicular components. This protocol describes a reproducible workflow for isolating sEVs from mouse SkM and BM. SkM is enzymatically digested, whereas BM is processed directly and subsequently subjected to differential centrifugation and size-exclusion chromatography (SEC). For SkM, the void volume (~2.5 mL) is discarded, and the subsequent ~1.6 mL is collected as EV-enriched fractions, typically subdivided into 400 &#181;L sequential fractions (F1&#8211;F4). For BM, a 700 &#181;L void volume is discarded, followed by collection of an ~850 &#181;L EV-enriched fraction, which can be subdivided into ~170 &#181;L sequential fractions (F1&#8211;F5) and pooled based on EV marker enrichment. Using this approach, sEVs can be isolated from small tissue volumes (approximately 500 &#181;L from a single quadriceps and 150 &#181;L from pooled femur and tibia BM), yielding ~108 particles&#183;mL&#8722;1&#183;mg&#8722;1 tissue with particle diameters predominantly &#60;200 nm. Vesicle integrity and purity are validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting for canonical EV markers, with additional characterization by single-particle interferometric detection to quantify tetraspanin-defined vesicle subpopulations. This method enables reproducible isolation of high-purity sEVs from structurally complex tissues using minimal input material, supporting downstream molecular and functional analyses of tissue-derived vesicles.

To assess the effectiveness of this protocol for isolating tissue-derived sEVs, representative results from SkM and BM samples are presented. Successful isolation was defined by (i) enrichment of canonical EV markers (TSG101, CD63, CD81, and CD9) in early SEC fractions, (ii) minimal detection of intracellular contaminants (e.g., Calnexin and BiP), (iii) a unimodal particle size distribution with the majority of particles &lt;200 nm, (iv) visualization of intact, membrane-bound vesicles by TEM, and (v) higher particle con...

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.

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, ...

isolation-small-extracellular-vesicles-from-murine-skeletal-muscle

Research

JoVE Journal

Biology

Source: Watson, D. C., et al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the use of size exclusion chromatography with porous resin beads to isolate bacterial extracellular vesicles (EVs) from a bacterial culture filtrate. Larger EVs elute early by bypassing the resin pores, while smaller proteins enter the pores and elute later.

Isolation of Bacterial Extracellular Vesicles Using Size-Exclusion Chromatography 

In this video, we demonstrate a technique–size exclusion chromatography–to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion Chromatography.

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

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

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

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

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles (SkM-EVs) from rodent muscle tissues through mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It demonstrates consistency and reliability. The isolated SkM-EVs provide insights into muscle homeostasis and diseases, with potential applications as diagnostic biomarkers or therapeutic vehicles.

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

Learn to isolate small extracellular vesicles from mouse skeletal muscle and bone marrow using size-exclusion chromatography. This protocol enables high-purity sEV isolation from minimal tissue input for downstream molecular and functional analyses.

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

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