Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry
Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry
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DOI：
10.1002/cyto.a.23767
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发表时间：
2019-05-01
期刊：
影响因子：
3.7
通讯作者：
Coban, Cevayir
中科院分区：
文献类型：
--
作者：
Lelliott, Patrick M.;Momota, Masatoshi;Coban, Cevayir
中性粒细胞胞外陷阱（NET）的形成涉及DNA在细胞外的释放以中和病原体。活体显微镜、流式细胞术和活体成像等技术可以表征NET，但这些技术要么不能在体内应用，要么缺乏特异性，要么需要侵入性程序。我们开发了一种自动化分析方法，使用成像流式细胞术快速获取和表征细胞作为NET或NET前体，而不是经历其他形式细胞死亡的细胞。NET保持在溶液中使用一种新的三维细胞培养系统，其中细胞悬浮在两种不同密度的液体的界面。至关重要的是，我们确定NET使用图像分析算法的基础上形态数据显示挤出的DNA超出细胞边界。在体外，我们使用这种技术来证明不同的要求NET形成在人类和小鼠中性粒细胞。我们还测量了小鼠感染约氏疟原虫期间全血中的NET。我们希望这项技术将提供一种有价值的方法，以更好地了解NET形成的过程及其在疾病中的重要性。(C)2019年国际细胞计数促进学会
Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three-dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. (C) 2019 International Society for Advancement of Cytometry