Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50–150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)‐labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence‐conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application‐specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

中文翻译：

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高分辨率成像流式细胞术揭示了孵育温度对用抗体标记胞外囊泡的影响。

细胞外囊泡 (EV) 基本上从所有细胞中释放出来。在过去的十年中，小型 EV（sEV；50-150 nm）在诊断和治疗中获得了极大的关注。然而，方法上的限制加上电动汽车标准的缺乏，在这个快速发展的领域中留下了许多悬而未决的问题。最近，通过使用增强型绿色荧光蛋白 (eGFP) 标记的 sEV 作为生物参考材料，我们系统地优化了用于单个 sEV 分析的成像流式细胞术。此外，我们表明可以以多参数方式分析用不同荧光抗体染色的 sEV。然而，许多可能影响 sEV 染色程序的参数仍需要进一步评估和优化。在这里，我们对孵化温度（4°C，室温和 37°C）在 sEV 抗体染色期间对涉及用荧光偶联抗体染色 EV 的实验结果。我们提供的证据表明，染色强度和样品回收率都可能因应用的孵育温度而异，并且在延长孵育时间后观察到的差异不太明显。此外，这项研究可以作为 EV 流式细胞术参数评估的特定应用示例。© 2020 作者。并且在延长孵育时间后观察到的差异不那么明显。此外，这项研究可以作为 EV 流式细胞术参数评估的特定应用示例。© 2020 作者。并且在延长孵育时间后观察到的差异不那么明显。此外，这项研究可以作为 EV 流式细胞术参数评估的特定应用示例。© 2020 作者。Cytometry Part A由 Wiley Periodicals, Inc. 代表 International Society for Advancement of Cytometry 出版。