A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation
A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation
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DOI：
10.3791/59150
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发表时间：
2019-01-01
影响因子：
1.2
通讯作者：
Teng, Y. K. Onno
中科院分区：
文献类型：
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作者：
Arends, Eline J.;van Dam, Laura S.;Teng, Y. K. Onno
中性粒细胞胞外陷阱 (NET) 是具有免疫原性的胞外 DNA 结构，中性粒细胞在多种触发因素下均可释放。 NETs 已被证明是一种重要的宿主防御机制，可以捕获和杀死微生物。另一方面，它们与多种系统性自身免疫性疾病有关。 NET 是免疫原性和毒性结构，含有一系列相关自身抗原，包括抗中性粒细胞胞浆抗体 (ANCA) 相关血管炎 (AAV) 和系统性红斑狼疮 (SLE)。根据刺激，可以诱导不同形式的 NET。 NET 的量可以使用不同的技术进行定量，包括测量上清液中的 DNA 释放、测量与髓过氧化物酶 (MPO) 或中性粒细胞弹性蛋白酶 (NE) 等 NET 分子复合的 DNA、通过荧光显微镜测量瓜氨酸组蛋白的存在或对 NET 成分进行流式细胞术检测，这些技术在特异性、敏感性、客观性和数量方面都具有不同的特征。这是一个使用三维免疫荧光共聚焦显微镜以高灵敏度、高通量方式量化离体 NET 形成的协议。该协议可用于解决有关健康和疾病中 NET 形成和退化的各种研究问题。
Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.