Leary Noelle, Walser Sarina, Dieterich Lothar C
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland.
Methods Mol Biol. 2022;2504:199-206. doi: 10.1007/978-1-0716-2341-1_14.
Extracellular vesicles (EVs), comprising exosomes, ectosomes, and apoptotic bodies, are an important component of molecular cell-to-cell communication, and are critically involved in the pathophysiology of various diseases, including tumors. In order to study the interaction of tumor cell-derived EVs with their target cells and to investigate their biological functions in comparison to other tumor cell-released factors, efficient isolation of EVs from cultured tumor cells, as well as fluorescent labeling of these EVs, is often necessary. In addition, EVs and EV-like particles are emerging as versatile vehicles for the delivery of therapeutic substances. Here, we describe an easy size exclusion chromatography-based method to isolate EVs from the mouse melanoma cell line B16F10 that yields highly enriched EV samples for subsequent applications such as molecular and functional studies. Our protocol also includes an optional labeling step with the lipophilic dye DiD, which allows tracking of EV uptake by recipient cells in vitro and in vivo.
细胞外囊泡（EVs）包括外泌体、微囊泡和凋亡小体，是细胞间分子通讯的重要组成部分，并在包括肿瘤在内的各种疾病的病理生理学中起关键作用。为了研究肿瘤细胞衍生的细胞外囊泡与其靶细胞的相互作用，并与其他肿瘤细胞释放的因子相比研究其生物学功能，通常需要从培养的肿瘤细胞中高效分离细胞外囊泡，以及对这些细胞外囊泡进行荧光标记。此外，细胞外囊泡和类细胞外囊泡颗粒正成为递送治疗物质的通用载体。在此，我们描述了一种基于尺寸排阻色谱的简便方法，用于从小鼠黑色素瘤细胞系B16F10中分离细胞外囊泡，该方法可产生高度富集的细胞外囊泡样品，用于后续的分子和功能研究等应用。我们的方案还包括用亲脂性染料DiD进行的可选标记步骤，这允许在体外和体内追踪受体细胞对细胞外囊泡的摄取。
