Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles.

IF 1.2 4区综合性期刊 Q3 MULTIDISCIPLINARY SCIENCES

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI:10.3791/67439

Yaochao Zheng, Aiden Charles Streleckis, Hongyu Chen, Yao Yao

{"title":"Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles.","authors":"Yaochao Zheng, Aiden Charles Streleckis, Hongyu Chen, Yao Yao","doi":"10.3791/67439","DOIUrl":null,"url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2000,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Jove-Journal of Visualized Experiments","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/67439","RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}

引用次数: 0

Abstract

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.

查看原文本刊更多论文

求助全文

约1分钟内获得全文求助全文

来源期刊

Jove-Journal of Visualized Experiments MULTIDISCIPLINARY SCIENCES-

CiteScore

2.10

自引率

0.00%

发文量

992

期刊介绍： JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.