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一种将钙黄绿素-AM加载到哺乳动物细胞外泌体中的方案，以便通过与反卷积系统耦合的荧光显微镜进行清晰观察。

A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.

作者信息

Calderón-Peláez María-Angélica, Castellanos Jaime E, Velandia-Romero Myriam L

机构信息

Virology Group, Vice-chancellor of Research, Universidad El Bosque, Bogotá, Colombia.

出版信息

PLoS One. 2025 Jan 24;20(1):e0317689. doi: 10.1371/journal.pone.0317689. eCollection 2025.

DOI:10.1371/journal.pone.0317689
PMID:39854328
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11761115/
Abstract

Extracellular vesicles (EVs) are membrane-bound structures produced and released into the extracellular space by all types of cells. Due to their characteristics, EVs play crucial roles in cellular communication and signaling, holding an immense potential as biomarkers and molecular transporters. Various methods have been developed to label and characterize EVs, however, visualizing EVs remains a process that requires highly specialized and expensive equipment, which is not always available in all the laboratories. In this study, we adapted a protocol originally designed for EVs analysis by flow cytometry using Calcein-AM, and convert it into a useful and effective tool for visualizing EVs by epifluorescence microscopy coupled with a deconvolution system. This approach can be very useful for basic EVs analyses, enabling researchers to verify their distribution and internalization across cells. Such insights can guide decisions on whether to advance to more detailed analysis using confocal microscopy or to perform additional assays.

摘要

细胞外囊泡（EVs）是由所有类型的细胞产生并释放到细胞外空间的膜结合结构。由于其特性，EVs在细胞通讯和信号传导中发挥着关键作用，作为生物标志物和分子转运体具有巨大潜力。人们已经开发出各种方法来标记和表征EVs，然而，可视化EVs仍然是一个需要高度专业化且昂贵设备的过程，而并非所有实验室都能随时获得这些设备。在本研究中，我们改编了一种最初设计用于通过流式细胞术使用钙黄绿素-AM分析EVs的方案，并将其转化为一种通过落射荧光显微镜结合去卷积系统可视化EVs的有用且有效的工具。这种方法对于基础的EVs分析非常有用，使研究人员能够验证它们在细胞间的分布和内化情况。这些见解可以指导关于是否推进到使用共聚焦显微镜进行更详细分析或进行其他检测的决策。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/d8febdb6601e/pone.0317689.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/ea754d90092d/pone.0317689.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/4b29c74e296d/pone.0317689.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/2647c08a05f8/pone.0317689.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/99c08160ca4d/pone.0317689.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/d8febdb6601e/pone.0317689.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/ea754d90092d/pone.0317689.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/4b29c74e296d/pone.0317689.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/2647c08a05f8/pone.0317689.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/99c08160ca4d/pone.0317689.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4631/11761115/d8febdb6601e/pone.0317689.g005.jpg

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本文引用的文献

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Factors to consider before choosing EV labeling method for fluorescence-based techniques.在为基于荧光的技术选择荧光标记方法之前需要考虑的因素。
Front Bioeng Biotechnol. 2024 Sep 18;12:1479516. doi: 10.3389/fbioe.2024.1479516. eCollection 2024.
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Low pinocytic brain endothelial cells primarily utilize membrane fusion to internalize extracellular vesicles.低胞饮作用的脑内皮细胞主要通过膜融合内化细胞外囊泡。
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Fourteen years of cellular deconvolution: methodology, applications, technical evaluation and outstanding challenges.
十四年的细胞去卷积：方法学、应用、技术评估和突出挑战。
Nucleic Acids Res. 2024 May 22;52(9):4761-4783. doi: 10.1093/nar/gkae267.
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Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies.外源性和内源性细胞外囊泡的标记策略及其在体外和体内功能研究中的应用。
Cell Commun Signal. 2024 Mar 9;22(1):171. doi: 10.1186/s12964-024-01548-3.
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Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.细胞外囊泡研究的最低信息要求（MISEV2023）：从基础到先进方法。
J Extracell Vesicles. 2024 Feb;13(2):e12404. doi: 10.1002/jev2.12404.
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Macrophages reprogramming driven by cancer-associated fibroblasts under FOLFIRINOX treatment correlates with shorter survival in pancreatic cancer.在 FOLFIRINOX 治疗下，癌症相关成纤维细胞驱动的巨噬细胞重编程与胰腺癌患者的生存时间更短相关。
Cell Commun Signal. 2024 Jan 2;22(1):1. doi: 10.1186/s12964-023-01388-7.
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