CPHEN-017:外周人类中性粒细胞上的中性粒细胞胞外捕获物 (NET) 的综合表型。

IF 2.5 4区 生物学 Q3 BIOCHEMICAL RESEARCH METHODS
Ceridwyn Jones, Anne La Flamme, Peter Larsen, Kathryn Hally
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引用次数: 0

摘要

随着最近发现中性粒细胞能够产生细胞外陷阱(NET),中性粒细胞作为感染和炎症的积极参与者日益受到重视。NETs的特点是从中性粒细胞中挤出的DNA和蛋白质组成的大型网状网络,人们对这些结构如何驱动人类疾病产生了浓厚的兴趣。要推进这一领域的研究,必须开发出量化 NETosis 的新工具。为此,我们开发了一种 7 标记流式细胞仪面板,用于分析体外刺激后人外周中性粒细胞和炎症条件下新鲜循环中性粒细胞的 NETosis。对从全血中分离出来的中性粒细胞进行了优化,并对新鲜中性粒细胞或用磷酸-12-肉豆蔻酸-13-醋酸酯(PMA)或离子霉素(两种已知的NET诱导激动剂)体外刺激的中性粒细胞进行了分析。中性粒细胞被鉴定为 SSChighFSChighCD15+CD66b+ 。对胺残基和 7-Aminoactinomycin D(7-AAD)(我们选择的 DNA 染料)呈阳性的中性粒细胞被视为坏死细胞(Zombie-NIR+7-AAD+),并从下游分析中剔除。排除 Zombie-NIR 和 7-AAD 阳性(Zombie-NIRdim7-AAD+)在此被用作中性粒细胞附属 DNA 的标记,这是 NET 的一个关键特征。利用两种 NET 相关蛋白--髓过氧化物酶(MPO)和中性粒细胞弹性蛋白酶(NE)--的存在来识别中性粒细胞附属 NET 事件(SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+)。我们还证明,NET 中性粒细胞表达瓜氨酸化组蛋白 H3(H3cit),体外 PMA 和离子霉素刺激可诱导其浓度依赖性,但经 DNase 处理后会被分解,而且在慢性和急性炎症中都存在。这种 7 色流式细胞仪面板为检测人体的 NETosis 提供了一种新工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils

CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSChighFSChighCD15+CD66b+. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR+7-AAD+) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIRdim7-AAD+) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins – myeloperoxidase (MPO) and neutrophil elastase (NE) – were utilized to identify neutrophil-appendant NET events (SSChighFSChighCD15+CD66b+Zombie NIRdim7-AAD+MPO+NE+). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.

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来源期刊
Cytometry Part A
Cytometry Part A 生物-生化研究方法
CiteScore
8.10
自引率
13.50%
发文量
183
审稿时长
4-8 weeks
期刊介绍: Cytometry Part A, the journal of quantitative single-cell analysis, features original research reports and reviews of innovative scientific studies employing quantitative single-cell measurement, separation, manipulation, and modeling techniques, as well as original articles on mechanisms of molecular and cellular functions obtained by cytometry techniques. The journal welcomes submissions from multiple research fields that fully embrace the study of the cytome: Biomedical Instrumentation Engineering Biophotonics Bioinformatics Cell Biology Computational Biology Data Science Immunology Parasitology Microbiology Neuroscience Cancer Stem Cells Tissue Regeneration.
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