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Development of a quantitative method to measure EV uptake

机译：开发一种定量方法来衡量电动汽车的摄取

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The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.
机译：细胞外囊泡（EVs）在医学上的巨大潜力值得对调节其掺入靶细胞的分子方面进行详细研究。但是，由于EV大小低于光学技术的分辨率极限，因此在现有技术中通常无法完全实现量化以及EV与靶细胞结合和摄取之间的区别。将人四跨膜蛋白CD9和CD63融合到双重EGFP-Renilla分裂标签上。通过Western印迹和荧光显微镜评估这些融合蛋白的亚细胞定位和掺入电动汽车。 EV的结合和摄取是使用经典的Renilla底物或具有细胞渗透性的底物进行测量的。用含有互补DSP1部分的EV孵育表达DSP2的靶细胞无法恢复荧光或萤光素酶活性。但是，使用携带完全重构的Dual-EGFP-Renilla蛋白和可渗透细胞的Renilla荧光素酶底物的EV，我们可以区分EV与摄取的结合。我们通过分析不同化学抑制剂的作用来提供系统概念的证明，证明该方法高度灵敏和定量，可以在高通量方案中进行动态跟踪，以揭示不同生物中电动汽车摄入的分子机制。系统。

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