Extracellular vesicles (EVs) play pivotal roles in intercellular communication and are implicated in numerous physiological and pathological processes. Here, we introduce a quantitative technique using total internal reflection fluorescence microscopy (TIRFm)-based single vesicle analysis (SVA) to assess EV aggregation, a critical factor influencing their biological functionality. Employing two-colored fluorescent recombinant EV mixtures, this method enables precise discrimination between aggregated and non-aggregated EVs. It allows for calculating an aggregation ratio from the colocalization of fluorescence signals. We evaluate the impact of isolation methods, storage conditions, and biochemical environments on EV aggregation, including salt and pH variations and the presence of antibodies. Additionally, we quantitatively assess the efficacy of aggregation removal techniques, revealing significant variability in removal methods depending on the type of aggregates. This analytical approach is expected to enhance our understanding of EV aggregation dynamics and set a new standard for the characterization and functional analysis of EVs.

中文翻译：

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通过单个囊泡分析评估细胞外囊泡聚集


细胞外囊泡 （EVs） 在细胞间通讯中起着关键作用，并与许多生理和病理过程有关。在这里，我们介绍了一种使用基于全内反射荧光显微镜 （TIRFm） 的单囊泡分析 （SVA） 的定量技术来评估 EV 聚集，这是影响其生物学功能的关键因素。该方法采用双色荧光重组 EV 混合物，能够精确区分聚集和非聚集的 EV。它允许根据荧光信号的共定位计算聚集比率。我们评估了分离方法、储存条件和生化环境对 EV 聚集的影响，包括盐和 pH 值的变化以及抗体的存在。此外，我们定量评估了聚集体去除技术的有效性，揭示了去除方法的显着差异，具体取决于聚集体的类型。这种分析方法有望增强我们对 EV 聚集动力学的理解，并为 EV 的表征和功能分析设定新标准。