Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
Methods Enzymol. 2020;645:55-78. doi: 10.1016/bs.mie.2020.05.013. Epub 2020 Jun 22.
Virtually all cells release extracellular vesicles (EVs) into their environment, such as exosomes and microvesicles. EVs can mediate intercellular communication processes in a targeted manner. Representing their cell of origin, EVs contain cell type specific signatures, qualifying them as a novel class of biomarkers. Furthermore, according to their tropism to certain target cells, EVs provide promising aspects to be used as drug delivery vehicles. Depending on their origin, certain EVs contain the potential to modulate physiological and pathophysiological processes. Although the EV field provides many interesting aspects, the methodology in EV research is limited. For now, EVs are mainly analyzed by nanoparticle tracking analysis and bulk molecular analysis, regularly Western Blot. These technologies cannot dissect the heterogeneity of EVs observed by electron microscopy (EM). Although EM technologies help to demonstrate the heterogeneity within EV samples, EM technologies are not appropriate to perform more complex and quantitative EV analyses. Flow cytometry (FCM) is a traditional method for dissecting the heterogeneity of given cell populations in a quantitative and complex manner. However, classical FCM regularly fails to detect objects in the size range of small EVs (sEVs) that typically is in the range between 70 and 150nm. Recently, we and others demonstrated the potential of imaging FCM for the analyses of small EVs at the single vesicle level. Here, at the example of sEVs harvested from supernatants of human mesenchymal stromal cells (MSCs), we share a protocol for studying the expression of the tetraspanins CD9, CD63 and CD81 on single EVs.
几乎所有细胞都会向其环境中释放细胞外囊泡（EVs），如外泌体和微泡。EVs 可以以靶向的方式介导细胞间通讯过程。代表其起源细胞，EVs 包含细胞类型特异性特征，使它们成为新型生物标志物。此外，根据它们对特定靶细胞的趋化性，EVs 提供了作为药物递送载体的有前途的方面。根据其起源，某些 EVs 具有调节生理和病理生理过程的潜力。尽管 EV 领域提供了许多有趣的方面，但 EV 研究中的方法学有限。目前，EVs 主要通过纳米颗粒跟踪分析和批量分子分析，定期进行 Western Blot 进行分析。这些技术无法剖析电子显微镜（EM）观察到的 EV 的异质性。尽管 EM 技术有助于证明 EV 样品内的异质性，但 EM 技术不适合进行更复杂和定量的 EV 分析。流式细胞术（FCM）是一种用于以定量和复杂方式剖析特定细胞群体异质性的传统方法。然而，经典的 FCM 通常无法检测到小 EV（sEVs）大小范围内的物体，sEVs 的大小通常在 70 至 150nm 之间。最近，我们和其他人证明了成像 FCM 在单囊泡水平分析小 EV 的潜力。在这里，以从人间质基质细胞（MSCs）上清液中收获的 sEVs 为例，我们共享了一种研究单 EV 上四跨膜蛋白 CD9、CD63 和 CD81 表达的方案。
