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50ml锥形管中，用等体积的无菌室温度的混合血3％葡聚糖-500（0.9％无菌盐水组成）轻轻翻转。留在室温下直立18-20分钟。的稻草色上层转移到一个新的试管，直至其不再能够不污染由下红色层收集。在日红细胞Ë原管可被丢弃。 </li><li>离心上清在500 xg离心在4℃下10分钟。抽出并丢弃上清液。手动重新悬浮在10毫升室温的无菌磷酸盐缓冲盐水（PBS）细胞沉淀。注意，涡旋不应该被用来在这个协议中的任何阶段重新悬浮中性粒细胞。手工绘制关闭，而不是浇灭上清还建议在整个协议以最大限度地提高细胞产量。 </li><li> 10毫升室温多聚蔗糖和泛影酸钠细胞分离溶液（ 例如 ，的Histopaque 1077）添加到50毫升锥形管中。小心层在细胞分离溶液中的含细胞溶液。注意这并没有平衡到室温可减少细胞产量，使用细胞分离溶液。 </li><li>离心在300×g离心在室温下与制动切断30分钟。 </li><li>嗜中性粒细胞是在梯度的最低层，抽出并丢弃UPPER 3层，血浆和血小板，单核细胞的窄的白色带和密度梯度介质的透明层的上层的构成。 </li><li>以溶解任何残留的红细胞，再悬浮在10毫升室温的水中的嗜中性粒细胞颗粒。 </li><li> 30秒后，通过加入10ml的无菌室温1.8％氯化钠恢复张力和轻轻地倒转混合。 </li><li>离心机在500×g离心在4℃下5分钟。抽出上清液并丢弃。 </li><li>如果丸粒仍然是红色，重复裂解步骤。如果颗粒是白色的（或者如果裂解已经执行两次），轻轻地在10ml无菌PBS重新悬浮细胞。 </li><li>计数使用自动计数器或血球细胞。典型产量是每毫升血液中的至少10 6个细胞。 </li><li>离心机在500×g离心在4℃下5分钟。抽出上清液并丢弃。 </li><li>重新悬浮细胞在无菌室温PBS中所需的浓度。佛r中的DNA释放测定在第2节以每毫升5×10 6个细胞中所述，重悬。 </li><li>如果需要的话，直接转移细胞的一小体积或使用cytocentrifuge到载玻片。允许细胞干燥并使用根据制造商的指令的快速固定和染色试剂盒染色。如果在显微镜下观察的嗜中性粒细胞分离已经成功，有核细胞的至少95％应是粒细胞（嗜中性粒细胞，嗜碱性粒细胞，嗜酸性粒细胞）以最小的红细胞的污染。 </li></ol> 3. DNA释放分析<ol><li>中性粒细胞后，立即隔离设立DNA释放试验。 </li><li>如果细胞聚集存在于原液通过光学显微镜检查，立即设置在测定前通过无菌70微米无菌过滤器通过细胞悬浮液。 </li><li>到无菌的96孔板的每个测试孔中，加入5×10 4个细胞/孔;激动剂和罗斯威尔公园备忘录不含酚红和补充有0.5％热灭活的胎牛血清100-200微升的最终体积里亚尔研究所-1640（RPMI）培养基。包括其中的中性粒细胞原液是由等体积的PBS替换每激动剂至少两个空白孔。注意，对于每个激动剂设置空白孔是在通过用荧光测定激动剂排除激动剂或干扰的DNA污染重要。 </li><li>孵育在39℃下，在二氧化碳（4％）培养箱中30分钟。 </li><li>在所需的浓度和细胞不可渗透的核酸染料添加激动剂。注意细胞不可渗透的染料的最佳浓度将不同的核酸染料之间变化。其中激动剂由等体积的培养基代替非刺激对照应包括在每个实验。 注意:所希望的是稀释在培养基中的类似体积的激动剂保持孔之间是一致的条件。 200最终也卷＆＃181; l已成功使用，如果这个被改变，以及量应保持在所有条件相同。佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）（最终浓度≥0.1微米）或血小板活化因子（PAF）（最终浓度≥31微米）可被用作阳性对照。激动剂应至少一式两份进行测定。 </li><li>孵育在39℃。用酶标仪在2小时后，或在所需的时间间隔测量荧光。需要注意的是最佳的时间间隔将与所用激动剂而变化。 注:波长将依赖于所用的染料。 </li><li>计算平均荧光之前减去空白荧光。 </li><li>允许个人之间以及板之间的比较，由非刺激孔的平均荧光除以刺激孔的平均荧光计算荧光的倍数变化。 </li></ol>

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<li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+kill+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li>
<li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+is+immunity+the+second+function+of+chromatin%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> J Cell Biol. 198 (5), 773-783 (2012).</li>
<li>Chuammitri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chicken+heterophil+extracellular+traps+%28HETs%29%3A+novel+defense+mechanism+of+chicken+heterophils%5BTitle%5D)+AND+%22Vet+Immunol+Immunopathol%22%5BJournal%5D)">Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.</a> Vet Immunol Immunopathol. 129 (1-2), 126-131 (2009).</li>
<li>Palić, D., Ostojić, J., Andreasen, C. B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fish+cast+NETs%3A+neutrophil+extracellular+traps+are+released+from+fish+neutrophils%5BTitle%5D)+AND+%22Dev+Comp+Immunol%22%5BJournal%5D)">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/pubmed?term=((Antimicrobial+histones+and+DNA+traps+in+invertebrate+immunity%3A+evidences+in+Crassostrea+gigas%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invertebrate+extracellular+phagocyte+traps+show+that+chromatin+is+an+ancient+defense+weapon%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Invertebrate extracellular phagocyte traps show that chromatin is an ancient defense weapon.</a> Nat Commun. 5, 4627(2014).</li>
<li>Kaplan, M. J., Radic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+double-edged+swords+of+innate+immunity%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Neutrophil extracellular traps: double-edged swords of innate immunity.</a> J Immunol. 189 (6), 2689-2695 (2012).</li>
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<li>Martinod, K., Wagner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombosis%3A+tangled+up+in+NETs%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Antibodies+from+patients+with+rheumatoid+arthritis+target+citrullinated+histone+4+contained+in+neutrophils+extracellular+traps%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">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>
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</ol>

作者什么都没有透露。

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><tbody><tr><td>Plastic Whole Blood tube with spray-coated K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD</td><td>367835</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Dextran-500</td><td>Accurate chemical and scientific corp.</td><td>AN228410</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ThermoFisher scientific</td><td>20012043</td><td></td></tr><tr><td>Fetal bovine calf serum, heat inactivated</td><td>ThermoFisher scientific</td><td>10100139</td><td></td></tr><tr><td>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><td>96 well flat bottomed sterile polystyrene plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Platelet activating factor</td><td>Sigma-Aldrich</td><td>P4904</td><td></td></tr><tr><td>SYTOX Green Nucleic Acid Stain</td><td>ThermoFisher scientific</td><td>S7020</td><td></td></tr><tr><td>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 ...

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Immunology and Infection

8.0K 次观看.  Iowa State University. 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。 

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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 ...

科研

免疫与感染

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

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 response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

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

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence 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 ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

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

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.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

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. ...

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

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

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 ...

使用双染料活细胞分析定量 NET 形成

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

作者聚焦：使用双色活细胞成像定量疾病中的中性粒细胞细胞外陷阱和药物筛选

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

用于筛选不同信号通路的中性粒细胞功能检测

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

作者聚焦:使用 NeutroFun 筛选方案平衡中性粒细胞功能评估的速度、成本和准确性 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

一套快速了解中性粒细胞功能的筛查技术

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

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嗜中性粒细胞胞外的陷阱：如何生成和可视化

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移

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

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

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

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

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

一套快速了解中性粒细胞功能的筛查技术

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

摘要

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嗜中性粒细胞胞外的陷阱：如何生成和可视化

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

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

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

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

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

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

实时高通量技术量化人中性粒细胞中性粒细胞胞外陷阱的形成

一套快速了解中性粒细胞功能的筛查技术

体外共生分析评估病原体诱导中性粒细胞跨上皮迁移