Name: visualizing dengue virus through alexa fluor labeling
Uploaded: 2011-07-09T12:00:00
Description: Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

这项工作已经由国家医学研究理事会，新加坡的资助。

1。登革热病毒的Alexa Fluor标签<ol><li>标记反应之前，净化与蔗糖垫层登革热病毒，并准备必要的试剂和设备，在协议中表示。 </li><li>准备新鲜0.2M碳酸氢钠缓冲液，pH值8.5（标签缓冲区），1.5M羟胺缓冲液，pH 8.3（停试剂），之前的标签和过滤0.2μm的注射器过滤器消毒。 </li><li>淡化约3X10 8斑形成单位（PFU）登革热病毒的纯化标签在2毫升管的缓冲1毫升。这可缩放比例为一批病毒标签。 </li><...

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	<li>Olenych, S. G., Claxton, N. S., Ottenberg, G. K. &. a. m. p. ;. a. m. p., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fluorescent+Protein+Color+Palette.">The Fluorescent Protein Color Palette.</a> Current Protocols in Cell Biology. 21.5, 1-34 (2007).</li><li>Panchuk-Voloshina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alexa+dyes,+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright,+photostable+conjugates.">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+use+of+fluorescent+protein+markers+in+living+cells.">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li><li>Kuhn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+dengue+virus:+implications+for+flavivirus+organization,+maturation,+and+fusion.">Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.</a> Cell. 108, 717-725 (2002).</li><li>Aguilar, J. S., Roy, D., Ghazal, P., Wagner, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+blocks+herpes+simplex+virus-1+productive+infection+in+vitro+acting+at+different+stages+with+positive+cooperativity.+Application+of+micro-array+analysis.">Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.</a> BMC Infect Dis. 2, 9-9 (2002).</li><li>Zhang, S. L., Tan, H. C., Hanson, B. J., Ooi, E. E. <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+method+for+Alexa+Fluor+dye+labelling+of+dengue+virus.">A simple method for Alexa Fluor dye labelling of dengue virus.</a> J Virol Methods. 167, 172-177 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Alexa Fluor Succinimidyl Esters (Molecular Probes. , (2009).</li><li>Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+proteins+stained+by+Alexa+dyes.">Analysis of proteins stained by Alexa dyes.</a> Electrophoresis. 25, 779-784 (2004).</li><li>Freistadt, M. S., Eberle, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+poliovirus+for+flow+cytometric+cell+surface+binding+studies.">Fluorescent poliovirus for flow cytometric cell surface binding studies.</a> J Virol Methods. 134, 1-7 (2006).</li></ol>

虽然本报告中使用AF594染料，荧光团的Alexa Fluor琥珀酰亚胺酯系列广泛提供类似的标签化学。这可能会延长超越成像标签的应用。作为替代估计可兴奋和流式细胞仪机检测荧光团的标签程度的共聚焦显微镜，流式细胞仪可用于。 的Alexa Fluor染料小分子的反应，与游离氨基酸组，主要是精氨酸和赖氨酸9，一般向外面临的蛋白质残留。在我们的实验室，100μm的登革热病毒?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>碳酸氢钠</td><td> Sigma - Aldrich公司</td><td> S6297 </td><td></td></tr><tr><td>胲</td><td> Sigma - Aldrich公司</td><td> 159417 </td><td></td></tr><tr><td>氢氧化钠</td><td>默克公司</td><td> 106498 </td><td></td></tr><tr><td> AF594琥珀酰亚胺酯</td><td>分子探针，Invitrogen公司</td><td> A20004 </td><td></td></tr><tr><td> PD - 10柱</td><td> GE医疗集团</td><td> 17-0851-01 </td><td></td></tr><tr><td> HEPES </td><td> Sigma - Aldrich公司</td><td> H6147 </td><td></td></tr><tr><td>氯化钠</td><td> Sigma - Aldrich公司</td><td> S3014 </td><td></td></tr><tr><td> EDTA </td><td> Sigma - Aldrich公司</td><td> E9884 </td><td></td></tr><tr><td> M - 199 </td><td> Invitrogen公司</td><td> 11150 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070.03 </td><td></td></tr><tr><td> 4孔板</td><td> NUNC </td><td> 176740 </td><td></td></tr><tr><td>盖玻片</td><td> Einst </td><td> 0111520 </td><td></td></tr><tr><td>显微镜幻灯片</td><td>赛欧品牌</td><td> 7105 </td><td></td></tr><tr><td> 3H5杂交瘤细胞</td><td> ATCC </td><td> HB46 </td><td></td></tr><tr><td> 10X PBS </td><td>第一基地</td><td> BUF - 2040 - 10X1L </td><td></td></tr><tr><td>皂素</td><td> Sigma - Aldrich公司</td><td> S4521 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A7906 </td><td></td></tr><tr><td>氯化镁</td><td> Sigma - Aldrich公司</td><td> M2670 </td><td></td></tr><tr><td>氯化钙</td><td> Sigma - Aldrich公司</td><td> C3306 </td><td></td></tr><tr><td> Paraformaldehye </td><td> Sigma - Aldrich公司</td><td> 15,812-7 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> Calbiochem </td><td> 475904 </td><td></td></tr><tr><td> DABCO </td><td> Sigma - Aldrich公司</td><td> D27802 </td><td></td></tr><tr><td>台式离心机</td><td> Eppendorf公司</td><td> 5424 </td><td></td></tr><tr><td>共聚焦显微镜</td><td>蔡司</td><td> LSM 710 </td><td></td></tr></tbody></table>要准备的M - 199生长的培养基，添加胎牛血清50ML，丙酮酸钠和非必需氨基酸，500毫升的M - 199，无菌过滤5毫升5毫升。 要准备的M - 199维修保养液，添加15毫升胎牛血清，丙酮酸钠和非必需氨基酸，500毫升的M - 199，无菌过滤5毫升5毫升。

visualizing dengue virus through alexa fluor labeling

感染的结果，在病毒与细胞之间的相互作用的早期事件，可以有深远的影响。确定这种相互作用的影响因素可能会导致改善对疾病发病机理的认识，从而影响疫苗或治疗方案设计。因此，发展的方法来探测这种互动将是有益的的。在荧光团的发展1-3和成像技术，4最新进展，可以被利用来提高我们现有的知识对登革热的发病机制，从而铺平了道路，以减少每年发生登革热感染数以百万计的。 笼罩登革热病毒有一个外部的脚手架90包膜糖蛋白（e）保护核衣壳外壳的二聚体，其中包含一个正链RNA基因组的5组成。病毒表面上的相同的蛋白质亚基，从而可以与胺反应染料标记，并通过免疫荧光显微镜观察。在这里，我们提出了一个标签的Alexa Fluor琥珀酰亚胺酯染料直接溶解，取得了高度可行的病毒后，标签中的碳酸氢钠缓冲登革热病毒的简单方法。没有标准化程序的活病毒和现有的制造商的协议，对蛋白质的标签，通常需要染料在二甲基亚砜重组标签。二甲基亚砜的存在，即使在微量，可以阻止生产的病毒感染，也能诱导细胞的细胞毒作用 6 。排除使用在本议定书中的二甲基亚砜，从而降低了这种可能性。的Alexa Fluor染料具有优异的耐光性和低于常见的染料，如荧光素和罗丹明2，pH敏感，使他们的病毒对细胞的吸收和内体运输研究的理想。的Alexa Fluor染料的共轭不影响病毒特异性抗体，并在宿主细胞内 7公认的受体的识别标记的登革热病毒。这种方法可以在病毒学研究中的有用的应用程序。

Watch this Scientific Journal Video about Visualizing Dengue Virus through Alexa Fluor Labeling at JoVE.com

Visualizing Dengue Virus through Alexa Fluor Labeling

利用荧光的发展和成像技术，简单的方法，设计可视化早期病毒与细胞之间的相互作用的病毒，登革热的Alexa Fluor标签的进步。

可视登革病毒通过的Alexa Fluor标签

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Research

JoVE Journal

Immunology and Infection

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

科研

免疫与感染

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

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Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

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 ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

实时定量聚合酶链反应检测感染细胞培养中的登革热病毒 rna

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

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Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

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The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...