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><li>重组冻干的Alexa Fluor 594标记缓冲液立即标记反应前的琥珀酰亚胺酯（AF594）到1mm。根据人的需要，可以使用的Alexa Fluor染料系列荧光。尽量减少暴露在光线下这一步起。 </li><li>枪头轻轻的搅拌，添加1MM AF594染料100μL稀释病毒。 </li><li>在室温下孵育标签在黑暗中反应1小时组合。由每15分钟的温和的倒置混合。 </li><li>在台式离心机旋转管简单，枪头轻轻的搅拌，添加停止试剂100μL反应混合物。 </li><li>在室温下孵育在黑暗中增加一小时。由每15分钟的温和的倒置混合。 </li></ol> 2。净化的Alexa Fluor标记的登革热病毒<ol><li>与此同时，平衡与选择的缓冲区纯化柱。在这个实验中，PD - 10柱25毫升HNE缓冲区（5MM HEPES，150mm的氯化钠，EDTA的0.1MM），pH值7.4，在使用前平衡。 </li><li>应用标记的病毒列的顶部，并开始收集流量通过标记的病毒一旦进入矩阵。 HNE缓冲区的填充柱，所有标记的​​病毒一旦进入矩阵。弃流过的第一个2.5毫升和收集的旁边标有"病毒的一小部分2毫升。 </li><li>分装和存储AF594标记登革热病毒纯化-80 ° C，远离光源。 </li></ol><ol><li>解冻之一等份，并确定使用前一批标有病毒斑块检测滴度病毒的标记。 </li><li>种子5X10 4 Vero细胞每口井的M - 199生长的培养基中生长，在盖玻片上以及感染前一天的底部的4孔板。 </li><li>删除培养上清100μL卷（中的M - 199维持液稀释）标记病毒的多重感染1感染的细胞10min后在37 ° C。 </li><li>删除的接种，并两次在1xPBS洗盖玻片。 </li><li>在30分钟的3％paraformalydehyde的修复细胞。 </li><li>洗净1xPBS盖玻片3次。 </li><li>通透通透的解决方案的细胞含有皂甙0.1％，并在30分钟1xPBS的5％BSA。 </li><li>孵育的细胞与未稀释的离心澄清3H5单克隆抗体杂交瘤细胞培养上清在潮湿室1小时，避光。 </li><li>在洗涤缓冲液清洗盖玻片3次（1xPBS含1mm的氯化钙，1毫米氯化镁和0.1％皂素）。 </li><li>孵育细胞与AF488抗鼠IgG抗体，1:100稀释，在通透的解决方案，避光45分钟，在潮湿的室内。 </li><li>在洗涤缓冲液清洗盖玻片3次，并在去离子水冲洗一次。 </li><li>民建联对纸巾排出多余的水分和8μlMowiol 4-88含2.5％的DABCO安装在载玻片，盖玻片边缘。 </li><li>允许使用蔡司共聚焦显微镜才能浏览安装的解决方案，定于4℃过夜。连续并购应进行激动人心的荧光基团，在时间和随之而来的探测器之间切换。 </li><li>图像，然后分析了合作本地化发送蛋白质与标记病毒的抗体，使用蔡司LSM的禅宗软件估计的标签程度的染色。 </li></ol> 3。代表性的成果 AF594染料标记的登革热病毒产量的一个例子是如图2所示。通常情况下，不到10倍，从最初的滴度下降，应遵守以下成功的标签。但是，应该指出的是，所有的缓冲区必须做好准备新鲜的标签获得成功的Alexa Fluor琥珀酰亚胺酯应该用于重建后立即，因为他们在8水溶液中发生反应的游离酸水解。 下一步要检查之前，在实验中使用足够的荧光标记病毒。做一个简单的免疫荧光法在Vero细胞和一定程度的标签，可以从病毒的标记与抗- E蛋白抗体染色的合作定位估计。断绝AL细胞进行了检查，在图3所示是一个典型的共聚焦图像。共定位分析证明重叠系数从0.65到0.8不等的LSM的禅宗软件的使用，这表明约65％至80％的病毒粒子与染料标记的图像。 <img src="/files/ftp_upload/3168/3168fig1.jpg" alt="图1" /> 图1.Overall计划描绘登革热病毒程序的Alexa Fluor染料标签 ，首先，有关缓冲区和登革热病毒纯化准备。重组的Alexa Fluor染料和添加标记缓冲液稀释到登革热病毒。反应，然后停止1个小时后，除了停止试剂。随后，纯化标记的病毒是通过体积排阻列删除免费染料。最后，标记的病毒重新滴​​定斑块的检测和荧光测试。 <img src="/files/ftp_upload/3168/3168fig2.jpg" alt="图2" /> 图2。 AF594标记的登革热病毒等分解冻，重新滴定斑块的检测和它通常小于10倍下降至平均斑块检测前后AF594标签上确定的可行的病毒粒子（PFU / ml）的数量。开始滴度。误差线表示标准偏差的重复。 <img src="/files/ftp_upload/3168/3168fig3.jpg" alt="图3" /> 图3。成长上盖玻片前一天AF594标签在Vero细胞与登革病毒E蛋白的共定位 。细胞与感染登革热AF594标记在教学语言为10分钟1在37 ° C 。随后的细胞固定和抗E抗体标记，和E蛋白（绿色）和AF594标签（红色）的共定位研究。下63X放大倍率，使用蔡司LSM 710共聚焦显微镜观察荧光信号。比例尺为10μm。黄色表示的共定位等领域，如插图所示。

<ol>
	<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的登革热病毒的Alexa Fluor 594染料的共轭成像提供足够的亮度与病毒滴度的损失降到最低。不同亮度，可实现不同浓度的染料。然而，染料浓度的增加可以减少病毒的生存能力 7,10 。一个可能的限制是在受体结合，由于封锁的荧光团访问干扰。因此，根据实际应用，标签的最佳水平来确定，以确保标签和功能abrogation10的程度之间的平衡。千万注意，浓度也被包装后的反应性影响的Alexa Fluor琥珀酰亚胺酯8。 与这里介绍的Alexa Fluor染料登革热病毒的直接标记不要求任何额外的标签步骤，以可视化的病毒，从而消除了间接抗病毒抗体的非特异性染色的可能性。它还可以进行实时跟踪在活细胞成像的内化后的事件。这种方法比较简单，因为共轭是稳定的，它可以用来反对lipophillic荧光染料，如长链carbocyanine1 ,1 -双十八烷基- 3，3，生产和储存多个实验批次标记病毒， 3,3 - tetramethylindodicarbocyanine（DID）或苯乙烯基染料，不能在寒冷的存放超过3天。

<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 ...

visualizing-dengue-virus-through-alexa-fluor-labeling

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.8K 次观看.  DSO National Laboratories. 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。

视频: Visualizing Dengue Virus through Alexa Fluor Labeling

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统:可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

科研

免疫与感染

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

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 ...

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

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 to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

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

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA 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

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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

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

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

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

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

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

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

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

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.