Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout—ExoPLA

Q1 Health Professions

Current Protocols in Cytometry Pub Date : 2017-07-05 DOI:10.1002/cpcy.22

Liza Löf, Linda Arngården, Tonge Ebai, Ulf Landegren, Ola Söderberg, Masood Kamali-Moghaddam

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引用次数: 7

Abstract

Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive diagnostic targets. However, detection and characterization of EVs is challenging due to their small size. We've established a method, called ExoPLA, that allows individual EVs to be detected and characterized at high specificity and sensitivity. Based on the in situ proximity ligation assay (in situ PLA), proximal oligonucleotide-conjugated antibodies bound to their targets on the surfaces of the EVs allow formation of circular products that can be fluorescently labeled by rolling circle amplification. The intense fluorescent signals produced in this assay allow detection and enumeration of individual EVs by flow cytometry. We describe the procedures for ExoPLA, along with expected results and troubleshooting. © 2017 by John Wiley & Sons, Inc.

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利用流式细胞术Readout-ExoPLA近距离结扎法检测细胞外囊泡

细胞外囊泡(EVs)由大多数细胞连续释放，它们携带其起源细胞的表面标记物。EVs存在于所有体液中，作为细胞信息的传递者，有证据表明它们是疾病的标志。这些特点使电动汽车成为有吸引力的诊断目标。然而，由于电动汽车体积小，其检测和表征具有挑战性。我们已经建立了一种名为ExoPLA的方法，可以以高特异性和灵敏度检测和表征单个电动汽车。基于原位接近结扎试验(原位PLA)，近端寡核苷酸偶联抗体与ev表面的靶标结合，形成圆形产物，可以通过滚动圈扩增进行荧光标记。在该试验中产生的强烈荧光信号允许通过流式细胞术检测和枚举单个ev。我们描述了ExoPLA的程序，以及预期的结果和故障排除。©2017 by John Wiley &儿子,Inc。

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来源期刊

Current Protocols in Cytometry Health Professions-Medical Laboratory Technology

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期刊介绍： Published in affiliation with the International Society for Advancement of Cytometry, Current Protocols in Cytometry is a "best practices" collection that distills and organizes the absolute latest techniques from the top cytometry labs and specialists worldwide. It is the most complete set of peer-reviewed protocols for flow and image cytometry available.