概要

عالية الإنتاجية قياس خارج الخلية DNA الإصدار والكمي NET تشكيل في العدلات الإنسان<em> في المختبر</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

وافق مجلس المراجعة المؤسسية للجامعة جورجيا دراسة موضوع الإنسان لجمع الدم المحيطي من المتطوعين الأصحاء (أوغ # 2012-10769-06). وقعت 5،7،8 متطوعي شكل الموافقة المسبقة المطلوبة قبل سحب الدم. البحث المنجز في هذه المقالة هو في الامتثال للمبادئ التوجيهية الأخلاقية للبحوث الطبية التي تج…

Representative Results

الأرقام في هذه المخطوطة تصف طريقة العزلة العدلة والإجراءات التجريبية، ونتائج ممثل الحالية مع شرح لتحليل البيانات. يبين الشكل 1 في خطوات متتابعة من إعداد العدلات البشري. ويمثل هذا البروتوكول واحد فقط طريقة ممكنة من العزلة العدلة. انه يعطي ك…

Discussion

تمثل المستجدة آلية جديدة رائعة التي العدلات قتل مسببات الأمراض. 1 على الرغم من أن الأدب والمستجدة تم التوسع بشكل مستمر على مدى السنوات العشر الماضية منذ اكتشافهم، عدة أسئلة هامة تتعلق دورها في علم الأحياء، وآلية وتنظيم لا تزال غير واضحة. المنهجية المناسبة لابد …

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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