概要

胞外DNA释放的高通量测量和人中性粒定量NET形成<em>体外</em

Published: June 18, 2016
doi:

概要

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Abstract

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation.

Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Introduction

NET formation is a novel mechanism by which neutrophils fight pathogens.1 The core of NETs is nuclear DNA.1 This DNA network is associated with neutrophil granule proteins and histones.1 The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.2 NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release.

Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6

Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

Protocol

佐治亚大学的机构审查委员会批准的人类主体研究,以收集来自健康志愿者(UGA#2012-10769-06)外周血。5,7,8志愿者签署抽血前所需的知情同意书。在这篇文章中进行的研究符合涉及赫尔辛基宣言人体医学研究的伦理准则。 1.人外周血中性粒细胞的分离(图1) 注意:有几种方法分离自外周血的中性粒细胞。以下方案提供了一种可能性。该协议产生大量能够对外界刺激释放教师的?…

Representative Results

在这个手稿附图描述嗜中性粒细胞分离,实验程序和数据分析的解释本代表性结果的方法。 图1示出人中性粒细胞的制备的顺序步骤。该协议仅表示嗜中性粒细胞分离的可能的方式。它会产生大量的休息能够刺激后释放的外籍英语教师的中性粒细胞。 图2显示了基于荧光的DNA释放试验是如何工作的。使用DNA释放的荧光酶标仪动力学在人嗜中性粒细?…

Discussion

英语教师代表是中性粒细胞杀死病原体。1虽然教师的文学过去十年里,因为他们发现,与他们的生物学,机制和调控作用,一些重要的问题仍不清楚不断扩大在一个迷人的新机制。适当的方法已被开发来衡量英语教师,这非常独特的抗菌机理。本文介绍了可用于定量英语教师在高通量的方式方法。第一测定如下的膜不透DNA结合染料的荧光的动力学。此法提供了大量的斜坡上,距离中性粒?…

開示

The authors have nothing to disclose.

Acknowledgements

Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

Materials

Anti-Human Neutrophil Elastase  Rabbit Ab  Calbiochem 481001 1:2000X coated
Anti-Myeloperoxidase Ab (Rabbit) Millipore 07-496 1:2000X coated
DNase-1 Roche 10-104-159-001 1ug/ml used for digestion
20mM EGTA/ PBS Sigma-Aldrich E3889-25G
2.5 mM EGTA/PBS Sigma-Aldrich E3889-25G
Cell death detection ELISA Anti-DNA POD Roche 11544675001 1:500X 
Eon Microplate Spectrophotometer Biotek
Gen5 All-in-One microplate software Biotek analytical tool (ELISA)
Sytox orange Life Technology S11368 0.2% final concentration/volume
1 M Hepes Cellgro 25-060-Cl Use 10 mM final concentration.
1 M glucose Sigma Use 5 mM final concentration.
HBSS Corning 21-023-CM
Varioskan Flash Ver.2.4.3 Thermoscientific
PMA Sigma P 8139 100nM final used
ELISA Plate Greiner bio-one 655061
Conical tubes 15ml Thermoscientific 339650
Conical tubes 50ml Thermoscientific 339652
Percoll (pH 8.5-9.5)  Sigma P 1644 Sodium Chloride, Sigma, S7653-250G
Dextran Spectrum D1004
RPMI 1640 media Corning Cellgro 17-105-CV
96 well assay plate black plate clear bottom Costar 3603

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記事を引用
Sil, P., Yoo, D., Floyd, M., Gingerich, A., Rada, B. High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro. J. Vis. Exp. (112), e52779, doi:10.3791/52779 (2016).

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