The Brady Urological Institute, Johns Hopkins University School of Medicine; Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST);
The Brady Urological Institute, Johns Hopkins University School of Medicine.
J Vis Exp. 2022 Feb 14(180). doi: 10.3791/62836.
There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.
需要实际的测定方法来可视化和定量细胞的细胞外囊泡（EV）摄取。EV 摄取在各个研究领域的细胞间通讯中起着重要作用；癌症生物学、神经科学和药物输送。文献中已经报道了许多 EV 摄取测定方法；然而，缺乏实用的、详细的实验方法学。可以通过荧光标记 EV 来检测其在细胞内的位置来评估 EV 摄取。区分细胞内内化的 EV 和细胞表面的 EV 对于准确确定 EV 摄取非常重要，然而，这是具有挑战性的。因此，本工作提出了一种通过三维（3D）荧光共焦显微镜高效定量 EV 摄取的测定方法。使用基于纳米过滤的微流控装置制备荧光标记的 EV，通过 3D 共焦显微镜进行可视化，然后通过先进的图像处理软件进行分析。该方案为在细胞水平上分析 EV 提供了一种稳健的方法学，并为高效分析提供了一种实用的方法。
