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首页> 外文期刊>Journal of Extracellular Vesicles >A novel multiplex bead-based platform highlights the diversity of extracellular vesicles
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A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

机译：一个新颖的基于多重珠的平台突出了细胞外囊泡的多样性

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The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.
机译：细胞外囊泡（EVs）的表面蛋白组成与原始细胞有关，可能在囊泡功能中起作用。由于分析小囊泡的技术挑战，单个电动汽车蛋白质含量的知识仍然有限。在这里，我们介绍了一种新颖的基于多重磁珠的平台，可在一个样品中研究多达39种不同的表面标记。捕获抗体珠与荧光标记的检测抗体的结合可分析带有两种抗体识别的表面标记的EV。这种新方法可以轻松筛选电动汽车群体上的表面标记。通过组合不同的捕获和检测抗体，可以获得有关相对表达水平和潜在囊泡亚群的其他信息。我们还建立了一个协议，通过受激发射损耗（STED）显微镜观察单个电动汽车。由此，可以通过荧光团偶联的抗体检测单个EV上的标记。我们使用多重平台和STED显微镜首次显示NK细胞衍生的EV和血小板衍生的EV分别不含CD9或CD81，并且从活化B细胞中分离出的EV包含不同的EV亚群。我们推测，根据我们的STED数据，四跨膜蛋白可能不是均匀分布的，但可能大多数以聚类的形式出现在EV亚群上。最后，我们证明了电动车混合物可以通过磁珠分离，并随后通过多重平台进行分析。基于多重磁珠的平台和STED显微镜都显示了电动汽车的亚群，到目前为止，大多数使用的分析工具都无法区分它们。我们希望深入了解电动汽车的异质性将有助于我们了解不同的电动汽车和功能。

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