Name: in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy
Uploaded: 2018-08-24T13:30:00
Description: Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

相应的作者由莫里斯动物基金会 (D15CA-907) 资助。这项研究得到了加州大学戴维斯分校马健康中心和同伴动物健康中心 (2016-24 F) 的资助。作者想感谢 Geena 的帮助与数字和尼格海谭德阮为她的视频协助。

此处描述的所有方法均经加州大学戴维斯分校机构动物护理和使用委员会批准 (协议编号: 18338)。
1. 收集血液
<ol><li>用注射器吸入21克针, 从头或颈静脉中抽出10毫升的血。</li>
	<li>为了避免过度的剪切应力, 在将血液转入含有肝素的管 (75 USP) 之前, 从注射器中取出针头。轻轻地将管子倒置几次, 以确保抗凝血和血液的充分混合。在将样品放在冰上之前, 检查是否有血块或红细胞...

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</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> Nature Reviews Microbiology. 5 (8), 577-582 (2007).</li><li>Kaplan, M. 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> The Journal of Immunology. 189 (6), 2689-2695 (2012).</li><li>Czaikoski, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Induce+Organ+Damage+during+Experimental+and+Clinical+Sepsis.">Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis.</a> PLoS One. 11 (2), e0148142 (2016).</li><li>Dwivedi, D. 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=Prognostic+utility+and+characterization+of+cell-free+DNA+in+patients+with+severe+sepsis.">Prognostic utility and characterization of cell-free DNA in patients with severe sepsis.</a> Critical Care. 16 (4), R151 (2012).</li><li>Mai, S. H., 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=Delayed+but+not+Early+Treatment+with+DNase+Reduces+Organ+Damage+and+Improves+Outcome+in+a+Murine+Model+of+Sepsis.">Delayed but not Early Treatment with DNase Reduces Organ Damage and Improves Outcome in a Murine Model of Sepsis.</a> Shock. 44 (2), 166-172 (2015).</li><li>Jeffery, U., 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=Dogs+cast+NETs+too:+Canine+neutrophil+extracellular+traps+in+health+and+immune-mediated+hemolytic+anemia.">Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.</a> Veterinary Immunology and Immunopathology. 168 (3-4), 262-268 (2015).</li><li>Letendre, J. A., Goggs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+cell-free+DNA+concentrations+in+dogs+with+sepsis,+trauma,+and+neoplasia.">Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.</a> Journal of Veterinary Emergency and Critical Care. 27 (3), 307-314 (2017).</li><li>Jeffery, U., Gray, R. D., LeVine, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Fluorescence+Assay+for+Quantification+of+Canine+Neutrophil+Extracellular+Trap+Release.">A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.</a> J Vis Exp. (117), (2016).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., 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:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> Journal of Visualized Experiments. (36), (2010).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> Journal of Visualized Experiments. (17), (2008).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+Blue+Exclusion+Test+of+Cell+Viability.">Trypan Blue Exclusion Test of Cell Viability.</a> Current Protocols in Immunology. 111, (2015).</li><li>Li, R. H. L., Ng, G., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+neutrophil+extracellular+trap+formation+in+canine+neutrophils+is+dependent+on+histone+H3+citrullination+by+peptidylarginine+deiminase.">Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase.</a> Veterinary Immunology and Immunopathology. 193, 29-37 (2017).</li><li>Masuda, S., 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=NETosis+markers:+Quest+for+specific,+objective,+and+quantitative+markers.">NETosis markers: Quest for specific, objective, and quantitative markers.</a> Clinica Chimica Acta. 459, 89-93 (2016).</li><li>Li, 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=PAD4+is+essential+for+antibacterial+innate+immunity+mediated+by+neutrophil+extracellular+traps.">PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.</a> Journal of Experimental Medicine. 207 (9), 1853-1862 (2010).</li><li>Leshner, M., 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=PAD4+mediated+histone+hypercitrullination+induces+heterochromatin+decondensation+and+chromatin+unfolding+to+form+neutrophil+extracellular+trap-like+structures.">PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures.</a> Frontiers in Immunology. 3, 307 (2012).</li><li>Luo, 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=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45 (39), 11727-11736 (2006).</li></ol>

我们在这里提出一个协议, 以观察细胞 permeant 染料和细胞 impermeant 染料的活犬中性粒细胞细胞核构象和 cfDNA 释放的变化。该方法的主要优点是, 它允许在无细胞固定的情况下, 通过高分辨率显微术实时检测网络形成, 为观察体外网状形成提供了一个简单而有价值的工具。由于这种化验不需要抗体检测的净成分, 它是理想的研究 NETosis 在物种特定的抗体有限的可用性。利用这一方法, 作者证明, ...

中性粒细胞是短寿命的颗粒, 负责初步防御入侵病原体。嗜中性粒细胞被招募到感染部位, 通过吞噬、脱粒和产生活性氧 (ROS)1消除微生物。在细菌或内毒素存在的情况下, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 由细胞外染色质修饰的组蛋白和颗粒蛋白, 如弹性蛋白酶和髓 (过氧化物酶)2。虽然蚊帐具有不可缺少的抗菌性能, 但越来越多的实验和临床证据表明, 脓毒症的过度过度网络形成可能导致多器官功能障碍和死亡3,4,5,6。
由于蚊帐可能在犬类中起到类似的病理生理作用, 预防或减少网状形成的治疗性干预可以作为脓毒症动物的新治疗策略。因此, 需要一种可靠的技术来评估和量化狗的 NETosis 和网络成分。包括无细胞 DNA (cfDNA) 和核在内的净成分在犬中性粒细胞和7、8、9的临床犬血浆中得到了评价。通过荧光化验, Goggs 和 Letendre 发现, 脓毒症犬的 cfDNA 水平比健康犬高8。虽然这些技术是高度客观和定量的, 测量 cfDNA 和核作为标记的 NETosis 是非特异的, 因为它们可以从坏死细胞以外的 NETosing 中性粒细胞。在这里, 我们描述了一种技术, 利用荧光显微镜检查活 NETosing 中性粒细胞的行为。我们还详细介绍了一种改进的协议, 使用双 immunolabeling 主观量化的网及其组成部分, 如髓内过氧化物酶和 citH3 在犬中性粒细胞10。

<table>
	<tbody>
		<tr>
			<td>Dextran from Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392</td>
			<td>Molecular weight 450,000 &#8211; 650,000</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Life Sciences</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>A1285801</td>
			<td>With divalent cations</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190136</td>
			<td>Without divalent cations</td>
		</tr>
		<tr>
			<td>E. coli O55:B5</td>
			<td>InvivoGen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S11368</td>
			<td>5 mM in DMSO; stains in cells with permeable membranes</td>
		</tr>
		<tr>
			<td>SYTO Green 16 Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7578</td>
			<td>1 mM in DMSO; stains in cells with intact membranes</td>
		</tr>
		<tr>
			<td>Surface-Amps NP-40</td>
			<td>Pierce</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslips</td>
			<td>neuVitro</td>
			<td>H-12-pdl</td>
			<td>12 mm diameter</td>
		</tr>
		<tr>
			<td>Anti-citrullinated histone H3 antibody</td>
			<td>Abcam</td>
			<td>Ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>Unconjugated goat anti-rabbit Fab fragments</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-007-003</td>
			<td>Specificity: IgG (H+L)</td>
		</tr>
		<tr>
			<td>Anti- Myeloperoxidase antibody</td>
			<td>Dako</td>
			<td>A0398</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-Diamidino-2-phenylin (DAPI)</td>
			<td>Life Technologies Corporation</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy

为了应对入侵的病原体, 中性粒细胞释放中性粒细胞胞外陷阱 (网), 它们是用组蛋白和抗菌蛋白修饰的 DNA 胞外网络。脓毒症期间过度的净形成 (NETosis) 和 citH3 释放与小鼠和人类多器官功能障碍和死亡有关, 但其对狗的影响是未知的。本文介绍了一种从全血中分离犬中性粒细胞的方法, 以观察和定量 NETosis。由葡聚糖沉淀产生的富含白细胞的等离子体, 通过商业上可用的密度梯度分离介质和细胞计数和生存能力测试收集的颗粒分离。为了观察实时 NETosis 在活中性粒细胞, 细胞 permeant 和细胞 impermeant 荧光核酸染色添加到中性粒细胞激活, 无论是由脂多糖 (LPS) 或佛波 12-酯 13-醋酸酯 (PMA)。随着时间的推移, 荧光显微镜观察到核形态和网状形成的变化。体外NETosis 进一步的特点是定位的无细胞 DNA (cfDNA), 髓 (过氧化物酶) 和瓜组蛋白 H3 (citH3) 使用改良的双 immunolabelling 协议。为了客观地量化网络形成和 citH3 表达, 使用荧光显微镜, 网和 citH3-positive 细胞是量化的盲目的方式使用可用的软件。该技术是评价犬中性粒细胞体外能力 NETosis 的一种特殊检测方法。

利用活体细胞成像协议, 研究人员可以观察细胞核形态、细胞膜完整性和 cfDNA 在活中性粒细胞中的存在。细胞 impermeant 核染料污渍细胞核酸红色的细胞与受损细胞膜。另一种细胞 permeant 染料, 标签细胞内的核酸在活细胞与完整的等离子膜。所有完整的中性粒细胞, 无论其治疗, 应出现绿色, 并表现出的特点肿块核在0到30分钟后刺激 (图 2A)。经治疗的中?...

Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

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

我们描述了从全血中分离犬中性粒细胞的方法, 用荧光显微镜观察活中性粒细胞的网状形成。还描述了使用免疫荧光显微镜定量分析网络形成和瓜组蛋白 H3 (citH3) 表达的协议。

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 ...

Research

JoVE Journal

Immunology and Infection

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

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 ...

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

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

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

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

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 ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

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

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

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 ...

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 ...