Name: high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro
Uploaded: 2016-06-18T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

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.

조지아 대학의 임상 시험 심사위원회는 건강한 지원자 (UGA 번호 2012-10769-06)에서 말초 혈액을 수집하는 인간 대상 연구를 승인했다. 5,7,8 자원 봉사자 혈액 그리기 전에 필요한 정보 동의서에 서명했다. 이 문서에서 수행 된 연구는 헬싱키 선언의 인체를 이용한 의학 연구에 대한 윤리 지침을 준수합니다. 주변 인간의 혈액에서 호중구의 1. 분리 (그림 1) 주 : 말초 혈액 호중구를 ?...

<ol>
	<li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li><li>Fuchs, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li><li>Kessenbrock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+neutrophils+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nat Med. 15, 623-625 (2009).</li><li>Leffler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+neutrophil+extracellular+traps+co-varies+with+disease+activity+in+patients+with+systemic+lupus+erythematosus.">Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus.</a> Arthritis Res Ther. 15, R34 (2013).</li><li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes.">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunol Lett. 160, 186-194 (2014).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation.">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184, 205-213 (2009).</li><li>Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudogout-associated+inflammatory+calcium+pyrophosphate+dihydrate+microcrystals+induce+formation+of+neutrophil+extracellular+traps.">Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.</a> J Immunol. 190, 6488-6500 (2013).</li><li>Yoo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+cystic+fibrosis+airway+inflammatory+markers+from+Pseudomonas+aeruginosa-stimulated+human+neutrophils+involves+NADPH+oxidase-dependent+extracellular+DNA+trap+formation.">Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.</a> J. Immunol. 192, 4728-4738 (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, ., Goosmann, U., Zychlinsky, C., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps,+how+to+generate+and+visualize+them.">Neutrophil extracellular traps, how to generate and visualize them.</a> J. Vis. Exp. , (2010).</li><li>Rada, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyocyanin-enhanced+neutrophil+extracellular+trap+formation+requires+the+NADPH+oxidase.">Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.</a> PLoS One. 8, e54205 (2013).</li><li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptide+LL-37+facilitates+the+formation+of+neutrophil+extracellular+traps.">The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.</a> Biochem J. 464, 3-11 (2014).</li><li>Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+DNA+in+the+serum+of+cancer+patients+and+the+effect+of+therapy.">Free DNA in the serum of cancer patients and the effect of therapy.</a> Cancer Res. 37, 646-650 (1977).</li><li>Raptis, L., Menard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+and+characterization+of+plasma+DNA+in+normals+and+patients+with+systemic+lupus+erythematosus.">Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.</a> J Clin Invest. 66, 1391-1399 (1980).</li><li>Chang, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+cell-free+serum+DNA+detected+in+patients+with+myocardial+infarction.">Elevated cell-free serum DNA detected in patients with myocardial infarction.</a> Clin Chim Acta. 327, 95-101 (2003).</li></ol>

그물 그물의 문학은 지속적으로, 생물학,기구 및 규제에서의 역할에 관한 몇 가지 중요한 질문이 불분명 남아 자신의 발견 이후 지난 10 년에 걸쳐 확대되고 있지만 병원균을 죽인다. 1 호중구하는 매혹적인 새로운 메커니즘을 나타냅니다. 적절한 방법은 그물이 매우 독특한 항균 메카니즘을 측정하기 위해 개발되어야한다. 이 문서는 높은 처리량 방식으로 그물을 정량하는 데 사용할 수?...

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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Anti-Human Neutrophil Elastase&#160; Rabbit Ab&#160;</td>
			<td>Calbiochem</td>
			<td>481001</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase Ab (Rabbit)</td>
			<td>Millipore</td>
			<td>07-496</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>DNase-1</td>
			<td>Roche</td>
			<td>10-104-159-001</td>
			<td>1ug/ml used for digestion</td>
		</tr>
		<tr>
			<td>20mM EGTA/ PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mM EGTA/PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell death detection ELISA Anti-DNA POD</td>
			<td>Roche</td>
			<td>11544675001</td>
			<td>1:500X&#160;</td>
		</tr>
		<tr>
			<td>Eon Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 All-in-One microplate software</td>
			<td>Biotek</td>
			<td></td>
			<td>analytical tool (ELISA)</td>
		</tr>
		<tr>
			<td>Sytox orange</td>
			<td>Life Technology</td>
			<td>S11368</td>
			<td>0.2% final concentration/volume</td>
		</tr>
		<tr>
			<td>1 M Hepes</td>
			<td>Cellgro</td>
			<td>25-060-Cl</td>
			<td>Use 10 mM final concentration.</td>
		</tr>
		<tr>
			<td>1 M glucose</td>
			<td>Sigma</td>
			<td></td>
			<td>Use 5 mM final concentration.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-023-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Ver.2.4.3</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P 8139</td>
			<td>100nM final used</td>
		</tr>
		<tr>
			<td>ELISA Plate</td>
			<td>Greiner bio-one</td>
			<td>655061</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 15ml</td>
			<td>Thermoscientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 50ml</td>
			<td>Thermoscientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (pH 8.5-9.5)&#160;</td>
			<td>Sigma</td>
			<td>P 1644</td>
			<td>Sodium Chloride, Sigma, S7653-250G</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Spectrum</td>
			<td>D1004</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Corning Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well assay plate black plate clear bottom</td>
			<td>Costar</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro

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.

이 원고의 수치. 호중구 격리, 실험 절차 및 데이터 분석의 설명과 함께 현재 대표적인 결과의 방법을 서술 한 인간 호중구 준비의 순차적 인 단계를 보여줍니다. 이 프로토콜은 호중구 격리의 하나의 가능한 방법을 나타냅니다. 그것은 자극에 그물을 방출 할 수있는 호중구 휴식의 많은 양을 산출한다. 형광 기반의 DNA 자료 분석의 작동 방식 <stron...

Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

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

세포 외 DNA 릴리스의 높은 처리량 측정 및 인간 호중구의 양적 NET 형성<em&gt; 체외</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Research

JoVE Journal

Immunology and Infection

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

2 광자 현미경을 사용하여 감염된 폐의 Neutrophils에 의해 Phagocytosis와 NET - 형성의 직접 관측

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

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방법은 지질 호중구의 변경 및 호중구 세포 외 트랩의 후속 형성을 연구하기 위해

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The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...