Name: high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro
Uploaded: 2016-06-18T13:00:00.000+00:00
Duration: 11 min 3 s
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.

佐治亚大学的机构审查委员会批准的人类主体研究，以收集来自健康志愿者（UGA＃2012-10769-06）外周血。5,7,8志愿者签署抽血前所需的知情同意书。在这篇文章中进行的研究符合涉及赫尔辛基宣言人体医学研究的伦理准则。 1.人外周血中性粒细胞的分离（图1） 注意:有几种方法分离自外周血的中性粒细胞。以下方案提供了一种可能性。该协议产生大量能够对外界刺激释放教师的非激活的人中性粒细胞。使用40毫升由静脉穿刺健康志愿者获得的全血。7,9 <ol><li>分装20毫升血液进入两个50毫升锥形管。加入10 mL 6％右旋糖酐。轻轻混匀。 </li><li>经过20分钟转移白细胞RICħ上层相而不采取任何沉淀的红细胞成清洁50ml锥形管中，并用无菌PBS填满它。 </li><li>离心机在400 xg离心10分钟。在4毫升重悬沉淀无菌PBS。 </li><li>制备85％的5个步骤的Percoll梯度-80％-75％-70％-65％在两个15毫升锥形管和层2毫升白细胞悬浮液在每个梯度的顶端。 </li><li>离心机800 XG与刹车关闭30分钟。 </li><li>收集在75％/ 80％的所有细胞（中性粒细胞）在所述层和70％/ 75％的界面。 </li><li>在PBS中洗涤细胞两次（350 XG，10分钟，RT）和使用血球计数细胞。 </li></ol> 2.使用微孔板荧光计外DNA释放的动力学测量注意:此方法能够在不可透过膜的DNA结合在96孔微孔板格式指示的DNA释放的染料（ 图2）的荧光变化的测量。 <ol><li>制备悬浮液在测定培养基嗜中性粒细胞（HBSS +1％（V / V）的自体血清+ 5mM葡萄糖）以2×10 6个细胞/ ml的浓度。 </li><li>添加5微摩尔/升的膜不可渗透的DNA结合染料。轻轻混匀。 </li><li>等分试样的人类嗜中性粒细胞（50μl/孔）到96孔黑色，透明底微板。确保测定培养基覆盖井的整个底部。如果没有，轻轻敲击侧面的板。 </li><li>预热10分钟的含板中性白细胞在37℃。 </li><li>在此期间，在它们的最终浓度的两倍制备刺激方案中使用（在37℃的温水测定培养基）。作为阳性NET控制使用人嗜中性粒用100nM佛波醇肉豆蔻乙酸酯（PMA）4小时触发最大净释放。-5,8- PMA刺激4小时，确保最大净释放而自发NET形成仍然很低5,8。 </li><li>制备微孔板荧光计进行测定（4小时，37℃，激发530nm处和用于发射590纳米）。&lt;/ LI&gt; <li>通过加入50μl的刺激溶液至50μl嗜中性粒细胞悬浮液刺激嗜中性粒细胞。使用0.5微克/毫升皂素于一体的测量最大DNA释放信号。 </li><li>将板阅读器，并测量每2分钟没有晃动的荧光变化4小时。 </li><li>进行数据分析，计算随时间的荧光归增加。 <ol><li>从端点值减去基线荧光所有井包括皂苷（ 图2A，B）。上升皂素荧光应该是最高的信号。它被称为"最大的DNA释放"（ 图2A）。 </li><li>由最大DNA释放除以未知样品的荧光增大计算未知样品中的DNA释放百分比。 </li><li>重复的平均结果之和作为"最大的DNA释放％"（ 图2C）。 </li></ol></li></ol> 3. NET表单的定量通过通货膨胀MPO-DNA和HNE-DNA化验ELISA 注意:在我们的实验室这些测定改性（MPO-DNA）或建立（HNE-DNA）通过测量MPO-DNA和HNE-DNA复合物的水平的定量NET形成5。 <ol><li>涂层的96孔高结合过夜捕捉抗体（50μl/孔）的能力的ELISA平板:抗MPO（1:2,000稀释度）或抗HNE（1:2,000）。 </li><li>洗涤ELISA板三次，用PBS（200μl/孔）并在室温用5％BSA和0.1％人血清白蛋白阻断它们为2小时（200μl/孔）。用PBS洗涤三次。 </li><li>在96孔微孔板以100,000个细胞/孔在100μl测定培养基的密度种子嗜中性粒细胞，并刺激网的形成。孵育细胞:4小时37℃。 </li><li>加入2 U / ml的DNA酶中性粒细胞上清液进行有限的DNA酶消化，以及混合。让他们在室温。 </li><li>通过加入11微升25mM的EGTA（最终浓度2.5毫摩尔）停止15分钟后的DNA酶。拌匀。苏收集pernatants干净的离心管。自旋样品在300 xg离心除掉细胞碎片在RT 5分钟。转让其上清液到干净的离心管。让他们在冰，直到准备。 </li><li>使用预先制备的"NET标准"的等分试样。 注意:NET-标准是由至少5个不同的，独立人供体获得的PMA刺激嗜中性粒细胞的DNA酶消化的上清液的混合。 <ol><li>要使用相同的标准很长一段时间，收集并从每个捐赠者分装大量中性粒细胞上清。收集例如2毫升PMA刺激嗜中性粒细胞的DNA酶消化的上清液将导致200等份（各10微升）足以为200 ELISA板。数据表征NET标准在图5中。 </li><li>刺激人中性粒细胞用100nM PMA 4小时，并应用DNA酶消化，以根据步骤3.4-3.5其上清液。 </li><li>收集，游泳池，分装（10微升）和自由泽（-20°C），中性粒细胞上清液。 </li><li>为了准备"网标"，从解冻至少五个独立的供体获得一个等份/供体，将它们混合，并保持他们在冰上。 </li><li>丢弃解冻的样品，并使用新鲜的下一个实验。 </li><li>准备1:PBS + EGTA网标准的2系列稀释。 </li></ol></li><li>稀释DNase1消化的样品（标准和未知上清液）20倍的PBS + EGTA和分装它们涂有捕获抗体ELISA板。 </li><li>孵育过夜ELISA板在4℃下，并用PBS洗三次。 </li><li>应用检测抗体溶液（100μl/孔）:辣根过氧化物酶缀合的抗DNA抗体（1:500，鼠标）。孵育在黑暗1小时。 </li><li>洗涤四次后，用PBS加TMB过氧化物酶底物:100微升/孔，30分钟。蓝颜色是过氧化物酶活性（母语存在）的指示。 </li><li>通过加入100μl/孔的1M HCl终止反应。该蓝色的解决方案会变成黄色（ 图5A）。 </li><li>在使用微板光度计450nm处读取吸光度。 </li><li>进行数据分析，计算相对于NET标准的MPO-DNA或HNE-DNA复合物的量。 <ol><li>减去从所有样品的测定培养基的背景光密度值。 </li><li>绘制针对其相对净含量（Y轴）（ 图5A）将稀释的标准样品的吸光度值（X轴）。 </li><li>使用该标准曲线的非饱和范围建立趋势线（采用最佳指数拟合从所使用的软件）（ 图5A）。这条趋势线的方程式决定OD值转化为定量母语的金额。 </li><li>插入未知数的测量OD值到从3.13.3趋势线方程，这将使未知样品为NET-标准（ 图5A）的百分比在教师的量。 </li><li>计算重复的（一式三份）平均值，并提交最终的数据（ 图5C）"MPO-DNA或HNE-DNA复合物（标准的百分比）的量。" </li></ol></li></ol>

<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. 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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. 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The authors have nothing to disclose.

英语教师代表是中性粒细胞杀死病原体。1虽然教师的文学过去十年里，因为他们发现，与他们的生物学，机制和调控作用，一些重要的问题仍不清楚不断扩大在一个迷人的新机制。适当的方法已被开发来衡量英语教师，这非常独特的抗菌机理。本文介绍了可用于定量英语教师在高通量的方式方法。第一测定如下的膜不透DNA结合染料的荧光的动力学。此法提供了大量的斜坡上，距离中性粒?...

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><tbody><tr><td>Anti-Human Neutrophil Elastase&amp;nbsp; Rabbit Ab&amp;nbsp;</td><td>Calbiochem</td><td>481001</td><td>1:2,000x coated</td></tr><tr><td>Anti-Myeloperoxidase Ab (Rabbit)</td><td>Millipore</td><td>07-496</td><td>1:2,000x coated</td></tr><tr><td>DNase-1</td><td>Roche</td><td>10-104-159-001</td><td>1 &amp;#181;g/ml used for digestion</td></tr><tr><td>20 mM 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&amp;nbsp;</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>100 nM final used</td></tr><tr><td>ELISA Plate</td><td>Greiner bio-one</td><td>655061</td><td></td></tr><tr><td>Conical tubes 15 ml</td><td>Thermoscientific</td><td>339650</td><td></td></tr><tr><td>Conical tubes 50 ml</td><td>Thermoscientific</td><td>339652</td><td></td></tr><tr><td>Percoll (pH 8.5-9.5)&amp;nbsp;</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.

在这个手稿附图描述嗜中性粒细胞分离，实验程序和数据分析的解释本代表性结果的方法。 图1示出人中性粒细胞的制备的顺序步骤。该协议仅表示嗜中性粒细胞分离的可能的方式。它会产生大量的休息能够刺激后释放的外籍英语教师的中性粒细胞。 图2显示了基于荧光的DNA释放试验是如何工作的。使用DNA释放的荧光酶标仪动力学在人嗜中性粒细?...

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.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

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

high-throughput-measurement-extracellular-dna-release-quantitative

Research

JoVE Journal

Immunology and Infection

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

13.0K 次观看.  University of Georgia. High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

视频: 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

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

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

科研

免疫与感染

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 ...

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 ...

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.

胞外DNA释放的高通量测量和人中性粒定量NET形成

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简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

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