Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry.

IF 4.2 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Nanomedicine : nanotechnology, biology, and medicine Pub Date : 2022-12-01 DOI:10.2139/ssrn.4240649

Liang Wu, W. Woud, C. Baan, D. Hesselink, E. van der Pol, G. Jenster, K. Boer

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引用次数: 3

Abstract

Urinary extracellular vesicles (uEVs) are promising biomarkers for various diseases. However, many tools measuring uEVs rely on time-consuming uEV isolation methods, which could induce sample bias. This study demonstrates the detection of single uEVs without isolation using imaging flow cytometry (IFCM). Unstained urine samples contained auto-fluorescent (A-F) particles when characterized with IFCM. Centrifugation successfully removed A-F particles from the unprocessed urine. Based on the disappearance of A-F particles, a gate was defined to distinguish uEVs from A-F particles. The final readouts of IFCM were verified as single EVs based on detergent treatment and serial dilutions. When developing this protocol to measure urine samples with abnormally high protein levels, 25 mg/mL dithiothreitol (DTT) showed improved uEV recovery over 200 mg/mL DTT. This study provides an isolation-free protocol using IFCM to quantify and phenotype single uEVs, eliminating the hindrance and influence of A-F particles, protein aggregates, and coincidence events.

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成像流式细胞术对单个尿细胞外囊泡的无隔离测定。

尿细胞外囊泡(uEVs)是一种很有前景的生物标志物。然而，许多测量uEV的工具依赖于耗时的uEV分离方法，这可能会导致样本偏差。本研究展示了成像流式细胞术(IFCM)在不分离的情况下检测单个uev。未染色的尿液样本含有自动荧光(A-F)颗粒，用IFCM进行表征。离心成功地从未处理的尿液中去除A-F颗粒。基于a - f粒子的消失，定义了一个栅极来区分uev和a - f粒子。根据洗涤剂处理和连续稀释，IFCM的最终读数被验证为单个ev。在制定该方案以测量异常高蛋白水平的尿液样本时，25 mg/mL二硫苏糖醇(DTT)比200 mg/mL DTT显示出更高的uEV回收率。本研究提供了一种无分离的方案，使用IFCM对单个uev进行量化和表型分析，消除了A-F颗粒、蛋白质聚集体和巧合事件的阻碍和影响。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Nanomedicine : nanotechnology, biology, and medicine 工程技术-纳米科技

CiteScore

11.10

自引率

0.00%

发文量

133

审稿时长

42 days

期刊介绍： The mission of Nanomedicine: Nanotechnology, Biology, and Medicine (Nanomedicine: NBM) is to promote the emerging interdisciplinary field of nanomedicine. Nanomedicine: NBM is an international, peer-reviewed journal presenting novel, significant, and interdisciplinary theoretical and experimental results related to nanoscience and nanotechnology in the life and health sciences. Content includes basic, translational, and clinical research addressing diagnosis, treatment, monitoring, prediction, and prevention of diseases.