1。从人体血液中的中性粒细胞的分离使用约24毫升EDTA或肝素（10 U / ml）的人体血液抗凝。 <ol><li>加入6毫升Histopaque 1119至15毫升猎鹰管和认真层之上的5至7毫升全血。 </li><li>离心20分钟800 XG没有刹车。 </li><li>吸丢弃黄色的和明确的顶层和转移较低偏红相食入猎鹰管粒。 </li><li>用PBS离心10分钟，在300 × G.填补猎鹰管清洗细胞</li><li>在此期间，准备用2 ml 10X PBS混合18毫升Percoll的一个100％的Percoll解决方案。随着1X PBS准备了4毫升的85％，80％，75％，70％和65％Percoll解决方案。 </li><li>准备用Percoll梯度分层2毫升每百分比递减的顺序彼此顶部2猎鹰管。 </li><li>离心后除去上清液，结合4毫升的PBS颗粒和悬浮沉淀细胞。 </li><li>仔细层2毫升的再悬浮到每个梯度。 </li><li>离心20分钟800 XG没有刹车。 </li><li>离心后除去顶层和大部分的65％，层与外周血单个核细胞，直到85％层收集到新的猎鹰管白色其余interphases。 </li><li>填补了10分钟，用PBS离心300 × G.猎鹰管清洗细胞</li><li>删除2毫升的PBS上清和悬浮沉淀细胞（通常&gt; 95％，中性粒细胞）。 </li><li>使用血球计数细胞。 </li></ol> 2。激活中性粒细胞<ol><li>准备一个24孔细胞培养板，每口井投入无菌的13毫米的圆形玻璃盖玻片（＃1,5） </li><li>种子2 × 10 5细胞500μL的RPMI（含2％的人血清白蛋白）每口井的CO 2培养箱1H在37 ° C. </li><li>同时准备600纳米的RPMI PMA的解决方案，并添加到100μL，每孔细胞。从15分钟可达4 h中的CO 2培养箱孵育37 ° C。 </li><li>在4％（最终浓度）的PBS溶解多聚甲醛固定细胞。 </li></ol>物业管理公司作为阳性对照，是迄今为止最有力的代理人诱导NET形成。另外，其他病原体的刺激或共同培育可用于NET的感应。 NET中形成的各自的地位，可以检查的时间当然是进行时，如果额外的平行样品准备。如果像Sytox绿色（Invitrogen）的非permeant DNA染料被添加到非固定细胞，只有细胞外的DNA会被检测出来。由于形成新的以英语为母语的英语教师以某种方式损害Sytox存在，每个时间点，一个平行的样品已被使用。 3。 NET通过免疫标记检测以英语为母语的英语教师是很脆弱的，即使固定后，非常小心，被操纵，否则多数人的意志得到失去了在准备。 <ol><li>小心地取出24孔培养板的贴壁细胞的玻璃盖玻片解除与弯针在边缘，并用细镊子抓住它。把盖玻片倒置的PBS下降。这是可以做到在封口膜覆盖试管架的板材。像这3次5分钟清洗。 </li><li>在同样的方式在下降0.5％Triton X - 100的的在室温下1分钟通透细胞孵育盖玻片。 3倍的PBS洗1分钟。 </li><li>准备与封口膜和湿纸巾的湿热试验箱。莱盖玻片倒置封闭液（5％驴血清）下降30分钟，在37 ° C。 </li><li>主要抗体，如MS反华宏和Rb抗中性粒细胞弹性蛋白酶，在封闭液稀释。 </li><li>传输覆盖在潮湿的室内单直接阻止缓冲区下降到初级抗体和孵育1 h后，于37 ° C。 </li><li> 5分钟，用PBS清洗3次。 </li><li>淡化二次抗体，如DK反MS CY2和DK反RB Cy3标记，在封闭液。 </li><li>传输覆盖到二级抗体下降到湿热试验箱单，孵育1 h后，于37 ° C。 </li><li> 5分钟，用PBS清洗3次。根据您的荧光显微镜，染色5分钟的DNA与赫斯特33342（1微克/毫升），这将需要紫外线激发或远红光激发Draq5洗两次与区。水。 </li><li>设置Mowiol20μL下降到载玻片和安装与细胞盖玻片倒置。细胞之间的盖玻片和幻灯片。 Mowiol下降，会形成一层薄薄的同质盖玻片厚度时定位是下降。通常情况下，有没有需要按样品。如果你想使用非浸入式镜头，试样准备进行检查。浸泡镜片的微观分析，让试样干燥约1小时，直到Mowiol凝固在试样边缘。 </li></ol> 4。准备以英语为母语的英语教师，扫描电子显微镜（SEM） <ol><li>邮政修复用2.5％戊二醛在24孔板上的玻璃盖单细胞。 </li><li>取出玻璃盖玻片含24孔培养板的细胞，并把一滴水倒挂。像这3次5分钟清洗。 </li><li>含有0.5％OsO4孵育30分钟到24孔细胞培养板盖玻片转移。 </li><li>取出玻璃盖玻片含24孔培养板的细胞，并把一滴水倒挂。像这3次5分钟清洗。 </li><li>盖玻片转移到24孔细胞培养板，其中包含30分钟的1％鞣酸和孵化。 </li><li>重复步骤4.2至4.4 </li><li>脱水通过以下治疗方案（5分钟）： <ul><li> 30％的乙醇</li><li> 50％的乙醇</li><li> 70％的乙醇</li><li> 80％的乙醇</li><li> 90％的乙醇</li><li> 100％的乙醇</li><li> 100％的乙醇</li><li> 100％的乙醇</li></ul></li><li>传输覆盖到临界点干燥机和干燥样品的单。 </li><li>涂层试样表面用一层薄薄的蒸发器与5nm的铂/碳层</li></ol> 5。代表性的成果： 隔离方法通常会产生未刺激可行的中性粒细胞纯度大于95％。当固定在不同时间点的刺激后，免疫协议显示NETosis细胞扁平，核小叶的损失，损失颗粒，细胞核完整，导致核（即组蛋白）和颗粒（即增加重叠期间的形态学变化序列中性粒细胞弹性蛋白酶）染色。这个协议可以作为一个出发点，以分析与中性粒细胞病原体的特异性相互作用。这种互动可以更用扫描电子显微镜的准备协议的详细解剖。 NET的荧光 NET组件（蓝色= DNA，红色=组蛋白，绿色=中性粒细胞弹性蛋白酶）染色的刺激中性粒细胞样本。图像显示，除了英语为母语的英语教师的定位，DNA和组蛋白的核本地化和中性粒细胞弹性蛋白酶颗粒格局。 教育统筹局局长PMA的刺激扫描电子显微照片显示非刺激的中性粒细胞和PMA刺激。刺激后，中性粒细胞的扁平化，生产以英语为母语的英语教师。 扫描电镜英语教师和志贺氏菌较高的分辨率的扫描电镜图像被困在以英语为母语的英语教师的痢疾杆菌。

<ol>
	<li>Brinkmann, V. <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 .</li><li>Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, 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+capture+and+kill+Candida+albicans+yeast+and+hyphal+forms.">Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.</a> Cell Microbiol. 8, 668-676 (2006).</li><li>M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+amazonensis+promastigotes+induce+and+are+killed+by+neutrophil+extracellular+traps.">Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.</a> PNAS. 106, 6748-6753 (2009).</li><li>Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase+expression+allows+the+pathogen+group+A+Streptococcus+to+escape+killing+in+neutrophil+extracellular+traps.">DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.</a> Curr Biol. 14, 396-400 (2006).</li><li>Fuchs, T. A. <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>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+suicide:+why+neutrophils+die+to+make+NETs.">Beneficial suicide: why neutrophils die to make NETs.</a> Nat Rev Microbiol. 5, 577-582 (2007).</li><li>Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+spontaneous+apoptosis+of+neutrophil+granulocytes+by+the+intracellular+parasite+Leishmania+major.">Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.</a> J Immunol. , 169-898 (2002).</li></ol>

所提供的协议将允许非刺激中性粒细胞纯度相当孤立，NET中形成的诱导和NETosis过程中的形态学变化的分析。当小心处理的标本，即避免了苛刻的洗涤条件，这将导致损失的最松散连接的以英语为母语的英语教师，NET不同的刺激条件下形成的量（持续时间，刺激）可以比拟的。在这方面，所提供的协议可以作为出发点，以建立的方法来分析更复杂的场景：中性粒细胞/病原体相互作用，刺激序列，与其他免疫细胞的相互作用。

neutrophil extracellular traps: how to generate and visualize them

嗜中性粒细胞粒细胞是最丰富的组的外周血白细胞。作为专业的吞噬细胞，吞噬细菌和细胞内杀死他们时，他们的抗菌颗粒吞噬的导火索。我们发现，嗜中性粒细胞杀死微生物的其他方式：激活后，他们释放颗粒蛋白和染色质，共同形成了外纤维结合的病原体。这些新颖的结构，或中性粒细胞（以英语为母语的英语教师）外的陷阱，降低致病因素， 并杀死1细菌，真菌和寄生虫3。结构以英语为母语的英语教师骨干的DNA，他们正在迅速退化DNases存在。因此，表达DNases的细菌毒性更强的 4 。使用相关显微镜，透射电子显微镜，扫描电镜，免疫荧光法和活细胞成像技术相结合，我们可以显示，刺激后，中性粒细胞的细胞核失去它们的形状和欧盟和异均质。后来，核膜和颗粒膜的瓦解使NET组件的混合。最后，以英语为母语的英语教师被释放细胞膜断裂。这种细胞死亡程序（NETosis）是有别于凋亡和坏死，取决于生成活性氧，NADPH氧化酶 5 。 在急性炎症的网站丰富的中性粒细胞外的陷阱。以英语为母语的英语教师似乎是先天免疫反应的一种形式，结合微生物，防止火势蔓延，并保证了很高的抗菌药物的局部浓度，降低致病因素，从而使中性粒细胞，以履行其抗菌功能，甚至超越了他们的寿命杀灭病原体。然而，越来越多的证据，以英语为母语的英语教师也参与疾病，自身免疫综合征不孕6。 我们描述的方法来隔离外围人体血液中7的嗜中性粒细胞，并刺激他们形成以英语为母语的英语教师。同时，我们也包括协议，在光学和电子显微镜可视化的以英语为母语的英语教师。

Watch this Scientific Journal Video about Neutrophil Extracellular Traps: How to Generate and Visualize Them at JoVE.com

Neutrophil Extracellular Traps: How to Generate and Visualize Them

中性粒细胞（以英语为母语的英语教师）外的陷阱是一个重要的先天免疫机制，对抗致病的细菌，真菌和寄生虫。在这里，我们描述的方法来隔离从人体血液中的中性粒细胞粒细胞和激活它们形成以英语为母语的英语教师。我们目前的制备技术，以可视化在光学显微镜和电子显微镜的以英语为母语的英语教师。

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 ...

neutrophil-extracellular-traps-how-to-generate-and-visualize-them

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

86.3K 次观看.  Max Planck Institute for Infection Biology. 中性粒细胞（以英语为母语的英语教师）外的陷阱是一个重要的先天免疫机制，对抗致病的细菌，真菌和寄生虫。在这里，我们描述的方法来隔离从人体血液中的中性粒细胞粒细胞和激活它们形成以英语为母语的英语教师。我们目前的制备技术，以可视化在光学显微镜和电子显微镜的以英语为母语的英语教师。

视频: Neutrophil Extracellular Traps: How to Generate and Visualize Them

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 ...

科研

免疫与感染

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

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.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

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 ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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. ...