转化科技UTMB-NIH授予1UL1RR029876，01和艾丽莎普拉瑟她的协助与视频编辑这篇稿子。

1。动物准备<ol><li>动物到达：到达后的动物生物安全2级（ABSL2）设施，让动物至少2天，新的环境适应。在此之后的休息时间，检查动物，以评估他们的健康和整体的物理外观。 <ol><li>利用荧光记者时，重要的是把所有的动物主题上苜蓿免费饮食，以限制GI量的自体荧光和背景信号。 </li></ol></li><li>剃须动物：为了提高从颅地区发现的生物发光信号，在成像过程中，所有的动物剃了头。麻醉的动物与氧气的混合物，并蒸发异氟醚。 ，一旦动物是完全麻醉，使用电推剪剃光头。一旦完成剃须，回到笼子里的动物，完全恢复的麻醉效果观察。 </li><li>生物军医数据系统（BMDS）转发器插入（后剃须的小鼠的动物完全麻醉）：对于微创的方法来跟踪温度的预​​编程的BMDS无线转发器，皮下植入每个鼠标的背部区域。这些转发器允许无线识别和温度记录。植入的应答之后，应用兽医级组织粘合剂到伤口上。 </li><li>基线集合：所有的动物，感染前，记录的基准温度和重量。采集血液的蚀斑减少中和试验（PRNT）分析，通过复古轨道（RO）出血，而老鼠是在麻醉状态下。采血后，申请兽用抗生素眼膏，以减少潜在的继发性细菌感染。考虑应该完成与此基准线集合动物被放置在麻醉下，由于应力对小鼠的数目的次数。未来的RO收藏小号hould完成后成像，而鼠标仍然是麻醉状态下。从RO的最大收集血液，应该是约200微升。 </li><li>感染：将小鼠麻醉下，如前面所述。接种通过鼻内途径，使用剂量为5×10 6至5×10 7 pfu的TC83-萤光素酶中的总体积为40微升稀释的磷酸缓冲盐水（PBS）中，通过鼻内感染。感染后，将老鼠他们的住房笼子内，观察复苏。 </li></ol> 2。动物影像 免责声明：在他们的住房单元，生物安全柜（BSC），或者的XIC遏制框在任何时候都养老鼠。此协议经批准的BSC是一类II型B1或B2。 <ol><li>注入所有动物用10微升的15毫克/毫升的浓度在每克体重腹腔内（IP）的萤光素。 <ol><li>小鼠接受Ampligen是injecteD IP剂量为12.5毫克/千克体重-4小时后感染或感染后48小时，根据他们的小组。 </li></ol></li><li>在注射之前，与荧光素，分离成三个一组的小鼠XIC-3遏制方块内，以确保一个适当的配合。 <ol><li>对于所有的成像，利用眼软膏/润滑剂，以防止整个手术过程中的干燥的角膜。 </li></ol></li><li>打开LivingImage软件，然后按初始化准备的成像盒，设置自动保存为自动，曝光时间（汽车的LivingImage软件3.2和更高版本的新功能，曝光时间），视场为C，打开过滤器;领域根据小鼠的数目，你是成像视图和过滤器可以被优化，如所需的其他成像项目。 </li><li>确认没有过期的空气活性炭过滤器，氧气罐是满的，充满异氟醚水库。在XIC-3我将一小条一小条的电工胶带溶胶箱，有助于减少动物的运动，在成像过程中。 </li><li>注入小鼠荧光素的IP在2.1节和地方3-5分钟内异氟醚诱导室（盖打开）。 </li><li> 5分钟后，关上盖子的感应室和异氟醚的流动。麻醉操作与氧流量设定在约3-5％，在约2.0升/分钟。一旦完全无意识再等待30秒，将小鼠的XIC-3的隔离箱。确保所使用的生物安全柜可清除异氟醚的安全，此过程将涉及损失的异氟醚的感应室（IVIS包含它自己的无源公平罐和XIC-3围堵箱/插座直接连接以确保载流）麻醉。 <ol><li>根据芯片/组号码的小鼠转移到XIC-3的异氟醚连接器连接的隔离箱D打开。位置的小鼠，使他们在胸骨斜卧头轻轻地搭在带。 </li></ol></li><li>用乙醇，喷雾的手和底部的XIC-3与cavicide擦拭XIC遏制框中，并转移到IVIS成像室。重新连接的麻醉遏制盒里面的成像单元。 </li><li> 10分钟后的萤光素的注射后，图像小鼠通过生物体的图像处理软件。 </li><li>在最初的影像以开放的过滤器，启动DLIT的3维成像，生物发光的萤火虫荧光素酶的序列分析和图像向导设置。这种成像过程将需要4-5分钟，并应完成的观点相关的所需的小鼠数目的字段。 <ol><li> 离体分析以下剖检和收集的大脑。将大脑在无菌培养皿中，滴50-100μl的股票萤光素之上，而在IMA晋框。 </li></ol></li><li>最后完成第二次公开滤波器图像DLIT成像完成后，在15-17分钟后感染。确保序列分析和领域的视图和过滤器设置恢复到以前的设置。 </li></ol> 3。返回老鼠和数据分析<ol><li>关闭异氟醚蒸发器和传输XIC遏制框生物安全柜。 </li><li>他们的住房单元内，将小鼠背部，合上，喷洒适当的消毒剂并放回外壳上的机架外部。 <ol><li>确保小鼠从麻醉中完全康复。 </li></ol></li><li>重复上述步骤，直到完成成像的实验需要。 </li><li>消毒BSC，关闭设备，将所有数据传输到外部实验室的位置，作进一步的分析。 </li><li>的数据进行分析，并生成IVIS结果利用率根据3维图像lizing LivingImage软件。 <ol><li>确保图像的正确导向，照明/色彩平衡设置。在工具选项板的选项包括影像调整，修正，图像信息，和投资回报率的工具。 </li><li>利用ROI的形状，以确定具体的基于位置的信号强度。 </li><li>突出的鼠身的表面形貌重建，初始化3D DLIT重建，并使用线性适合器官地图，地形重建。 </li></ol></li><li>通过收集的血液和器官，就可以完成进一步的病毒滴定分析。在这项研究中的滴定完成通过同质化改性的鹰培养基（MEM）中的脑组织。将匀浆液连续稀释，我们感染6孔板Vero细胞1小时。将细胞用1.5％的agarose/2xMEM重叠覆盖，在37℃孵育2天。 30分钟，用10％福尔马林固定细胞，染色结晶紫。 </li></ol>

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	<li>Steele, K. E., 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=Comparative+Neurovirulence+and+Tissue+Tropism+of+Wild-type+and+Attenuated+Strains+of+Venezuelan+Equine+Encephalitis+Virus+Administered+by+Aerosol+in+C3H/HeN+and+BALB/c+Mice.">Comparative Neurovirulence and Tissue Tropism of Wild-type and Attenuated Strains of Venezuelan Equine Encephalitis Virus Administered by Aerosol in C3H/HeN and BALB/c Mice.</a> Veterinary Pathology Online. 35, 386-397 (1998).</li><li>Ludwig, G. 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=Comparative+neurovirulence+of+attenuated+and+non-attenuated+strains+of+Venezuelan+equine+encephalitis+virus+in+mice.">Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.</a> Am. J. Trop. Med. Hyg. 64, 49-55 (2001).</li><li>Charles, P. C., Walters, E., Margolis, F., Johnston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Neuroinvasion+of+Venezuelan+Equine+Encephalitis+Virus+in+the+Mouse.">Mechanism of Neuroinvasion of Venezuelan Equine Encephalitis Virus in the Mouse.</a> Virology. , 208-662 (1995).</li><li>Volkova, E., Gorchakov, R., Frolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficient+packaging+of+Venezuelan+equine+encephalitis+virus-specific+RNAs+into+viral+particles+is+determined+by+nsP1-3+synthesis.">The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1-3 synthesis.</a> Virology. 344, 315-327 (2006).</li><li>Patterson, 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=Rapid,+non-invasive+imaging+of+alphaviral+brain+infection:+Reducing+animal+numbers+and+morbidity+to+identify+efficacy+of+potential+vaccines+and+antivirals.">Rapid, non-invasive imaging of alphaviral brain infection: Reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.</a> Vaccine. 29, 9345-9351 (2011).</li><li>Cook, S. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Monkeypox+Viruses+Pathogenesis+in+Mice+by+In+Vivo+Imaging.">Comparison of Monkeypox Viruses Pathogenesis in Mice by In Vivo Imaging.</a> PLoS ONE. 4, e6592 (2009).</li><li>Luker, G. D., Prior, J. L., Song, J., Pica, C. M., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+Reveals+Systemic+Dissemination+of+Herpes+Simplex+Virus+Type+1+in+the+Absence+of+Interferon+Receptors.">Bioluminescence Imaging Reveals Systemic Dissemination of Herpes Simplex Virus Type 1 in the Absence of Interferon Receptors.</a> J. Virol. 77, 11082-11093 (2003).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Kuehne, R. W., Pannier, W. L., Stephen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+mouse+model+for+the+evaluation+of+potential+antiviral+compounds:+results+with+Venezuelan+equine+encephalomyelitis+virus.">Indirect mouse model for the evaluation of potential antiviral compounds: results with Venezuelan equine encephalomyelitis virus.</a> Antimicrob. Agents Chemother. 11, (1977).</li><li>Lukaszewski, R. A., Brooks, T. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pegylated+Alpha+Interferon+Is+an+Effective+Treatment+for+Virulent+Venezuelan+Equine+Encephalitis+Virus+and+Has+Profound+Effects+on+the+Host+Immune+Response+to+Infection.">Pegylated Alpha Interferon Is an Effective Treatment for Virulent Venezuelan Equine Encephalitis Virus and Has Profound Effects on the Host Immune Response to Infection.</a> J. Virol. 74, 5006-5015 (2000).</li></ol>

没有利益冲突的声明。

虽然该协议涵盖了成像方面， 在体内分析，为今后的研究中的一个关键因素，重要的是要认识到生物发光的矢量。我们利用TC83，作为表达的荧光素酶的载体的减毒疫苗株VEEV确保正在生产大量的酶，由于如先前描述的1-4在中枢神经系统中的病毒的高复制率。虽然除了一个第二次基因组启动子和荧光素酶基因的重组病毒进一步衰减结果相比，野生型TC83，这种衰减不出现感染5?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> D-荧光素</td><td></td><td></td><td></td></tr><tr><td>异氟醚</td><td></td><td></td><td></td></tr><tr><td>精诺真IVIS系统（频谱） </td><td>卡尺生命科学</td><td></td><td></td></tr><tr><td> XGI-8气体​​麻醉系统</td><td>卡尺生命科学</td><td></td><td></td></tr><tr><td> XIC-3围堵箱</td><td>卡尺生命科学</td><td></td><td></td></tr><tr><td> LivingImage 4.0软件</td><td>卡尺生命科学</td><td></td><td></td></tr><tr><td>遥测/认筹</td><td>生物军医数据系统</td><td> IPTT-300 </td><td>动物ID和回火ATURE </td></tr><tr><td> BD的INTEGRA1毫升TB 26 GX 3/8“针注射器</td><td> Fisher Scientific则</td><td> 305279 </td><td></td></tr><tr><td>兽医债券组织粘合剂</td><td> Fisher Scientific则</td><td> NC9259532 </td><td></td></tr><tr><td> Vetropolycin眼药膏</td><td>韦伯斯特兽医产品</td><td> 78444656 </td><td></td></tr><tr><td> Dulbecco氏磷酸盐缓冲盐水1X </td><td> Invitrogen公司</td><td> 14190-144 </td><td></td></tr><tr><td> BMDS芯片阅读器</td><td>生物军医数据系统</td><td> DAS-7007S </td><td></td></tr><tr><td> DAS-HOST软件</td><td>生物军医数据系统</td><td></td><td>用于下载的探测信息</td></tr></tbody></table>

in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus

现代影像技术的进步，鼓励进一步发展和完善的方式来完成病毒的研究。最初提出的罗素和伯奇在休谟的3R原则（替代，减少，细化），利用动物模型，在科学研究上不断的压力，寻找新的方法来减少动物的使用量，同时提高科学性，准确性和速度。然而，休谟的校长面临的一个主要挑战是如何确保统计准确，同时减少动物疾病的发病率和总人数的研究。目前疫苗的药效学研究需要大量的动物才能被认为有统计学显着性，往往导致高发病率和高死亡率的端点识别的免疫保护。我们利用在体内成像系统（IVIS）一并萤火虫生物发光酶逐步跟踪由ENCE的中枢神经系统（CNS）的入侵小鼠模型中phalitic病毒。通常情况下，疾病的进展相对缓慢，但病毒复制是快速的，尤其是在中枢神经系统内，并且可以导致一个经常，致命的结果。 TC83 - 吕克·鼻内感染的小鼠后，委内瑞拉马脑炎病毒株的减毒修改，以表达荧光素酶基因，我们能够可视化的​​大脑内的病毒复制前至少3天的临床疾病症状的发展。脑炎病的一个关键发展端点，我们能够迅速地确定治疗和疫苗的保护，对TC83 - 吕克感染临床症状之前，利用CNS入侵的。通过IVIS技术，我们是能够表现出快速，准确的检测，药物治疗和疫苗，同时减少动物的数量和发病率。

的基因修饰的病毒，TC83荧光素酶，我们看到在生物发光信号强度的增加，作为病毒的复制移动到中央的中枢神经系统（ 图1），从鼻区域。由于高的病毒的复制率，我们期望看到的生物发光信号（ 图2A）的高浓度的取决于载体和动物的免疫反应的载体。我们预计这个信号的增加继续到了顶峰，感染后5-7天之间，在中枢神经系统内的病毒复制高峰（ 图2B）的组?...

Watch this Scientific Journal Video about In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus at JoVE.com

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

利用荧光素酶和体内成像系统（IVIS）作为一种新型的装置，以确定疾病临床发展发生之前的端点。 IVIS使我们能够实时的可视化入侵的脑炎病毒多天，为今后的研究提供了更精确的疾病模型。它也使我们能够找出潜在的抗病毒药物和疫苗的保护功能，速度比目前使用的动物模型。利用动物个体在多个时间点的能力，确保减少动物的要求，成本和总体发病率疾病的研究更人性化，更科学的方法确保使用的动物。

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

in-vivo-imaging-systems-ivis-detection-neuro-invasive-encephalitic

Research

JoVE Journal

Immunology and Infection

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

20.5K 次观看.  University of Texas Medical Branch. 利用荧光素酶和体内成像系统（IVIS）作为一种新型的装置，以确定疾病临床发展发生之前的端点。 IVIS使我们能够实时的可视化入侵的脑炎病毒多天，为今后的研究提供了更精确的疾病模型。它也使我们能够找出潜在的抗病毒药物和疫苗的保护功能，速度比目前使用的动物模型。利用动物个体在多个时间点的能力，确保减少动物的要求，成本和总体发病率疾?...

视频: In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

估计在水生系统的病毒产率

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...