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

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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.

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酶分离骨骼肌间质细胞外囊泡

细胞外囊泡（EVs）是由细胞释放的脂质双层封闭纳米颗粒，用于运输生物活性货物，如蛋白质、rna和dna，用于细胞间通讯。研究ev介导的肌肉内稳态和疾病中细胞间的串扰为我们理解肌肉的发育、再生和萎缩提供了巨大的潜力。然而，目前分离骨骼肌源性电动汽车（skm - ev）的方案在实现高纯度和产量方面面临挑战，主要是因为难以在不损害细胞膜的情况下从肌肉组织中释放电动汽车。本文提出了一种有效的SkM-EV分离方案，包括机械分离，酶解，过滤和超离心。这些步骤经过优化，以提高肌肉组织的EV释放，产生高纯度的skm -EV。随后，采用纳米流式细胞术、BCA检测和Western blot检测分离的skm - ev的数量和质量。该方案有望建立一个可靠的平台来获得组织源性电动汽车，以推进基础研究、疾病诊断和药物输送。

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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.