Name: a simple fluorescence assay for quantification of canine neutrophil extracellular trap release
Uploaded: 2016-11-21T14:00:00
Description: Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.

所有实验均来自爱荷华州立大学机构动物护理和使用委员会批准伦理进行。 1.采血车<ol><li>绘制的9ml血从隐，头或颈静脉直接进入抗凝剂（ 例如，乙二胺四乙酸（EDTA），1.8毫克的 K 2 EDTA /毫升血）真空采血管，或与直接传送到含有EDTA的血液管的注射器。轻轻摇动或反转血管，以确保血液和抗凝血剂的充分混合。 </li></ol> 2.中性粒细胞隔离<ol><li>在5...

<ol>
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B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cast+NETs:+neutrophil+extracellular+traps+are+released+from+fish+neutrophils.">Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.</a> Dev Comp Immunol. 31 (8), 805-816 (2007).</li><li>Poirier, A. C., Schmitt, P., Rosa, R. D., Vanhove, A. S., Kieffer-Jaquinod, S., Rubio, T. 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=Antimicrobial+histones+and+DNA+traps+in+invertebrate+immunity:+evidences+in+Crassostrea+gigas.">Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.</a> J Biol Chem. 289 (36), 24821-24831 (2014).</li><li>Robb, C. T., Dyrynda, E. A., Gray, R. D., Rossi, A. G., Smith, V. 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J., Radic, M. <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:+double-edged+swords+of+innate+immunity.">Neutrophil extracellular traps: double-edged swords of innate immunity.</a> J Immunol. 189 (6), 2689-2695 (2012).</li><li>Menegazzi, R., Decleva, E., Dri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+by+neutrophil+extracellular+traps:+fact+or+folklore?.">Killing by neutrophil extracellular traps: fact or folklore?.</a> Blood. 119 (5), 1214-1216 (2012).</li><li>Parker, H., Albrett, A. M., Kettle, A. J., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+associated+with+neutrophil+extracellular+traps+is+active+and+mediates+bacterial+killing+in+the+presence+of+hydrogen+peroxide.">Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide.</a> J Leukoc Biol. 91 (3), 369-376 (2012).</li><li>Martinod, K., Wagner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis:+tangled+up+in+NETs.">Thrombosis: tangled up in NETs.</a> Blood. 123 (18), 2768-2776 (2014).</li><li>Pratesi, F., 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=Antibodies+from+patients+with+rheumatoid+arthritis+target+citrullinated+histone+4+contained+in+neutrophils+extracellular+traps.">Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.</a> Ann Rheum Dis. 73 (7), 1414-1422 (2014).</li><li>Nauseef, W. 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提出的DNA释放法是外DNA一个容易量化分析。该方法改编自用于评估其他物种NET形成类似的技术，但离心速度，激动剂浓度和温育时间已被改变，以优化使用的方法与犬粒细胞8,17,18。类似的调整可以作出适应其他物种的方法。当与其他方法测量NETosis如流式细胞术或图像分析相比，该法是简单又便宜。该方法也并不需要对.NET组件抗体，所以非常适合在狗使用为谁商业化生产的，经过验证的?...

有超过7000万只宠物狗在美国独自1。为尊贵的家族成员，这些动物经常收到尖端的医疗护理。同样，因为他们分享我们的环境，狗可以提供深入了解人类疾病1的发病机理和治疗。然而，无论是在人类医学成兽医治疗或反之亦然翻译的发现，它彻底表征即使在高度保守的系统种类的变化，例如先天免疫应答是重要的。的犬和人先天免疫系统之间的差异例子包括中性粒细胞的狗2高表达CD4;缺乏胞质鞭毛传感器IPAF犬3功能同系物的和在食肉动物4胱天蛋白酶1/4混合的表达。 中性粒细胞胞外陷阱（英语教师）是先天免疫5的相对最近发现的组成部分。篮网是网络S IN响应释放到大范围炎性或传染性刺激6的DNA的核和颗粒状蛋白质。网状结构已被证实在许多物种包括鸡7，鱼8，软体动物9和acoelomates 10，但也有物种变体。例如，嗜中性粒细胞的小鼠更响应缓慢NETosis刺激比人类嗜中性粒细胞，形成弥漫性减弱英语教师11。有一个庞大的身躯从多个物种蚊帐坑害微生物和更具争议可以直接参与杀灭病原体12,13证据。然而，.NET组件也增强了组织的损伤，促进血栓形成，并作为自身抗原14,15。教师的有益和有害作用之间的平衡可能不同的疾病和不同物种之间变化，这表明，调查无论是在物种和感兴趣条件母语是重要的。 在这里，我们描述了诱导和中性粒细胞犬测量母语释放一个简单的协议。此方法类似于那些用于分离嗜中性粒细胞16和在其他物种中诱发NETosis，但如激动剂浓度和温育时间条件已为犬嗜中性粒细胞进行了优化。类似的NET定量的DNA释放测定也已在其它物种中描述，但这里介绍的方法也为狗8,17,18优化。

<table border="0" cellpadding="0" cellspacing="0" width="546">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:204px;">Plastic Whole Blood tube with spray-coated K2EDTA</td>
			<td style="width:215px;">BD</td>
			<td style="width:64px;">367835</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran-500</td>
			<td>Accurate chemical and scientific corp.</td>
			<td>AN228410</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ThermoFisher scientific</td>
			<td>20012043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine calf serum, heat inactivated</td>
			<td>ThermoFisher scientific</td>
			<td>10100139</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Media 1640, without phenol red or L-glutamine</td>
			<td>ThermoFisher scientific</td>
			<td>32404-014</td>
			<td>Should be free from phenol red</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat bottomed sterile polystyrene plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet activating factor</td>
			<td>Sigma-Aldrich</td>
			<td>P4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYTOX Green Nucleic Acid Stain</td>
			<td>ThermoFisher scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Synergy 2 Multi-Mode Reader</td>
			<td>BioTek</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple fluorescence assay for quantification of canine neutrophil extracellular trap release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

使用该协议，应该有在荧光与阳性对照，PMA和PAF刺激嗜中性粒细胞后强烈的倍数变化。如在图1B中 ，犬嗜中性粒细胞的刺激所示用31微米的PAF 1小时的结果，平均4.0倍的增加的荧光与未刺激细胞（范围2.0-5.8中，n = 5只犬）18进行比较。 PMA在0.1微米（ 图1A）是较慢激动剂不导致荧光的增加，直到2小时，但最终会产生荧光的类似倍的增加（平?...

Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

中性粒细胞胞外陷阱（英语教师）是DNA，组蛋白和中性粒细胞的蛋白质网络。虽然先天免疫反应的一个组成部分，母语有牵连的自身免疫和血栓形成。这个协议描述了犬嗜中性粒细胞的分离和使用微板荧光测定教师的量化的简单方法。

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Research

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Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

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Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

分化人类单细胞衍生巨噬细胞中细胞外陷阱释放的体外刺激与可视化

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

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Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

使用改良的夹心 ELISA 定量来自中性粒细胞胞外陷阱的髓过氧化物酶 DNA 和中性粒细胞弹性蛋白酶 DNA 复合物

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...