Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States.
Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States.
Methods Enzymol. 2020;645:1-14. doi: 10.1016/bs.mie.2020.02.001. Epub 2020 Feb 26.
Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.
分子成像方法是深入了解活细胞细胞组织的有力工具。为了了解细胞外囊泡 (EVs) 的生物发生和摄取，以及为靶向药物输送和治疗工程细胞衍生的囊泡，遗传标记与荧光蛋白已越来越多地用于确定 EVs 的结构、位置和动力学在体外和体内。在这里，我们报告了一种用于稳定标记 EV 的遗传方法，以研究它们在活的人类细胞中的生物发生和摄取。通过将绿色荧光蛋白 (GFP) 与内源性 CD63 (CD63-GFP) 或水疱性口炎病毒包膜糖蛋白 VSVG (VSVG-GFP) 融合，我们在细胞质中成功获得了不同的荧光信号，揭示了转染后细胞中 EV 的生物发生。我们详细描述了使用共聚焦显微镜和流式细胞术进行 EV 分离、成像和细胞摄取的实验程序。我们还提供了遗传标记方法如何用于研究 EV 生物学、工程 EV 表征和基于 EV 的纳米医学发展的观点。
