Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.

1，样品制备<ol><li>得到含有荧光素酶表达的病毒（本文VSVΔ51-Fluc为例）为滴定并转移至96孔板的样品。交替地，执行在96孔组织培养板直接与使用上清液感染。 </li><li>留下两列未处理列入标准曲线。用于实验的实验（40小时后的感染）或存储在-80℃和滴度的末端在96孔板中，滴度直接做在稍后的日期。 </li></ol> 2，准备允许细胞进行病毒滴定<ol><li> 24小时前滴定，制备Vero细胞的悬浮液以2.5×10 5个细胞/在Dulbecco改良的Eagle培养基毫升（DMEM）中含有10％胎牛血清（FBS），30mM的HEPES的浓度，和1％青霉素/链霉素。注：尽管本协议使用Vero细胞，任何适当的宽容细胞系的VSV感染可以bê使用。 </li><li>在使用微板的分配器96孔白色固体的平底板种子2.5×10 4个细胞（100微升）。 <ol><li>制备12毫升细胞悬浮液，每片加5毫升涂刷。 </li><li>干净微分配器盒通过冲洗管路用50ml无菌水。 </li><li>填充微分配器线路用细胞悬浮液，并让5毫升细胞悬浮液的流过。 </li><li>选择一个程序，将分配100μl的在白壁96孔板的每个孔中。分配，并重复所必需的附加板。还种子几孔中96孔透明平底板如果不透明底白墙板用于验证的细胞的健康和密度。 </li><li>完成后，冲洗细胞放回原包装内。通过运行50毫升70％乙醇中的随后加入50毫升的温无菌水通过管道清洁盒。 </li><li>确保磁带和油管是AP使用之间propriately清洗。 </li></ol></li><li>培养在湿润的5％CO 2培养箱中的细胞24小时，在37℃。 </li></ol> 3，病毒的制备标准曲线<ol><li>制备VSVΔ51-Fluc在无血清DMEM中的标准曲线，使得噬斑形成单位（pfu）每毫升转印后的最终浓度到Vero细胞上是如下：10 8 PFU / ml的10 7 PFU / ml的， 10 6 PFU / ml的10 5 PFU / ml的10 4 PFU / ml的，10 3 PFU /毫升，10 2 PFU / ml，并将10 1 PFU / ml的。准备50微升每盘Vero细胞上加上一个额外的10％，每个浓度。 请注意：在荧光素酶的病毒原液的效价将需要在一个经典方式10为了产生标准曲线，将允许为绝对定量评估。否则，相对定量可以在不准确的效价信息通过任意设定病毒滴度达到基础在稀释步骤。例如在标准曲线的第一稀释度可以被设置为10 8病毒单元和以下10稀释到10 7等。在这种情况下，应该表示的值作为倍数变化相比预先确定的样本作为绝对定量将不准确。 </li></ol> 4，样品转移上清到允许细胞<ol><li>检查Vero细胞接种在透明底的96孔板，用光学显微镜来确认单层是至少95％汇合。 </li><li>转账25微升样品上清液到Vero细胞接种于白墙板。不转移上清液到指定的标准曲线2列。注意：此可同时对所有的孔上使用96通道的液体处理的单个板来完成。 </li><li>每种稀释液的标准曲线的制备在步骤3到Vero细胞中的使用8位或12通道多通道移液器，加25微升2指定列。 </li><li>离心平板5分钟，在430 XG在RT。 </li><li>在潮湿的5％CO 2的培养箱中孵育5小时，在37℃。 </li></ol>细胞活性的5评价<ol><li>请注意：当从在清晰的96孔板进行感染实验中获得的上清液开始，样品活力之前可以量化与细胞存活率指示剂染料诸如刃天青评价。 </li><li>的量等于该体积的10％，在96孔板中含有病毒和细胞样品的每个孔中加入刃天青。包括细胞只控制以及媒体只控制分别确定为100％和0％生存力的值。 </li><li>后2-4小时（温育时间将取决于细胞类型和对染料的市售的或再造的粉末的浓度变化），读取并使用荧光板读数器（530-560激发，590发射）记录该信号。报告细胞viabili×100％根据下式的相对代谢活性= - （ - 负控制信号）/（细胞只能控制信号的负控制信号）（试验样品信号）TY为样品</li></ol> 6，制备荧光素底物<ol><li> 30分钟5小时孵育结束前，制备荧光素，以获得2毫克/毫升的溶液在无菌磷酸盐缓冲盐水（PBS）中。准备每盘2.5毫升加上一个额外的2毫升避光的解决方案。注：荧光素，也可以在当天早些时候制备并保存于4℃直至使用。 </li></ol> 7，读生物发光<ol><li> 5小时培养结束后，在白色的固体平板添加25μl的荧光素对Vero细胞的每个孔中。手动或使用自动分配器集成在光度计中添加荧光素。注意：只有单和终点使用的仪器读取可能需要添加荧光素酶裂解兼容的BUFFER之前加入萤光素底物，以改善一致性。 </li><li>阅读以下参数的板： <ol><li>摇动5秒。 </li><li>等待30秒。 </li><li>阅读发光在适当的固定感光度/曝光值。如果该选项可用在仪器上，使用多点读取（ 例如，3×3矩阵），以提高准确性。 </li></ol></li><li>记录并保存定量生物发光强度。 </li><li>如果一个自动化分配器用于添加荧光素清除萤光素和素用温水无菌水的线条。 </li></ol> 8，数据分析<ol><li>为准确的结果，解决了非线性回归，以产生从标准曲线希尔方程。套用这个公式的滴定样品来计算病毒表达单位。某些病毒或情况可能会产生的标准曲线的线性关系;在这种情况下求解线性方程。 </li></ol>

<ol>
	<li>Grigorov, B., Rabilloud, J., Lawrence, P., Gerlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+titration+of+measles+and+other+viruses:+optimization+with+determination+of+replication+cycle+length.">Rapid titration of measles and other viruses: optimization with determination of replication cycle length.</a> PLoS One. 6 (e24135), (2011).</li><li>Lizee, 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=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum Gene Ther. 14, 497-507 (2003).</li><li>McSharry, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+of+flow+cytometry+in+virology.">Uses of flow cytometry in virology.</a> Clin Microbiol Rev. 7, 576-604 (1994).</li><li>Watcharatanyatip, 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=Multispecies+detection+of+antibodies+to+influenza+A+viruses+by+a+double-antigen+sandwich+ELISA.">Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.</a> J Virol Methods. 163, 238-243 (2010).</li><li>Dawson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Quantification+of+Viruses+in+Solution+Using+the+ViroCyt+Virus+Counter.">Rapid, Direct Quantification of Viruses in Solution Using the ViroCyt Virus Counter.</a> Journal of Biomolecular Techniques. 23 (S10), (2012).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+and+RT-PCR:+true+or+false.">AAV and RT-PCR: true or false.</a> Mol Ther. 1, 389-390 (2000).</li><li>Diallo, J. S., Roy, D., Abdelbary, H., De Silva, N., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+infection+of+live+tissue+with+oncolytic+viruses.">Ex vivo infection of live tissue with oncolytic viruses.</a> J Vis Exp. 52, (2011).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl Microbiol. 16, 588-594 (1968).</li><li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adenoviruses:+propagation,+purification,+quantification,+and+storage.">Human adenoviruses: propagation, purification, quantification, and storage.</a> Curr Protoc Microbiol. 14 (14C 11), (2006).</li><li>Diallo, J. S., Vaha-Koskela, M., Le Boeuf, F., Bell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+purification,+and+in+vivo+testing+of+oncolytic+vesicular+stomatitis+virus+strains.">Propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains.</a> Methods Mol Biol. 797, 127-140 (2012).</li><li>Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ebola+virus+expressing+luciferase+allows+for+rapid+and+quantitative+testing+of+antivirals.">A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals.</a> Antiviral Res. 99, 207-213 (2013).</li><li>Diallo, J. 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=A+high-throughput+pharmacoviral+approach+identifies+novel+oncolytic+virus+sensitizers.">A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.</a> Mol Ther. 18, 1123-1129 (2010).</li></ol>

The authors have nothing to disclose.

这里描述的荧光素酶为基础的方法提供了许多优于其它现有的方法，包括其方便，快速，最小的设备的需要，并且成本相对较低。一个关键因素，这是避免连续稀释步骤。尽管如此，这个协议的系列稀释基衍生物是肯定可行的，最近被用于评估在高通量抗病毒药屏幕11荧光素酶表达埃博拉滴度。而本质上更耗时且较昂贵，这样的修改可以针对病毒的定量提供更大的动态范围，必要时。除了是特别适合用于评估病毒滴度在高通量筛选的上下文中，我们的一步法荧光素酶基的病毒定量方法产生在复制病毒的情况下，感染性病毒粒子的准确估计。此外，发光以及来自同一实验的细胞毒性数据读数得到更的靶细胞上的实验条件下的效果完整图像，这是在溶瘤病毒和药物屏幕的背景下尤其有用。 这里所示的例子中使用了复制的负单链RNA病毒;然而，该协议可以适应许多复制型或非复制型病毒有一些小的协议的调整。这包括DNA病毒，如牛痘，单纯疱疹病毒，和腺相关病毒（参见图5）。感染细胞内的病毒，如牛痘病毒，例如样品，需要事先向量化（ 例如 ，至少一个冷冻-解冻周期）的病毒的释放步骤。当应用该技术对其他的病毒，它是必要的，以从病毒上清液或裂解物由光度计转移到所述板的读取优化的温育时间。该参数将主要取决于病毒在允许细胞系和复制周期p的强度romoter驾驶荧光素酶的表达。这最好通过使用完整的标准曲线，在优化步骤中完成的。要做到这一点，必须有感染的编制标准曲线的各种重复的适当宽松细胞系和阅读每个重复在不同的时间点后转移。理想情况下，孵育时间点被选择，导致LOG（RLU）和LOG（效价）之间的线性关系跨越预期样品的滴度范围。为VSVΔ51-Fluc，这通常是从10 4 PFU / ml的-10 7 PFU / ml的为5小时的温育时间。如果更低或更高的滴度从样品预期，可以简单地增加或减少培养时间分别。或者，样品可以稀释到下降多达通常用于ELISA进行的范围内。 如上面所提到的，这种方法非常适合于执行使用药物库的大多数步骤可以是自动化的高通量药物屏幕。细胞可以被镀èfficiently使用自动化微板的分配器，所述药物库可使用96通道的液体处理器加入，病毒可以使用微分配器被添加和平板阅读使用自动化的光度计。从理论上讲，这也适用于384孔或更小的格式;但是，不限于该端是多个小区可以被镀的，给予较少的细胞导致更窄的范围中的LOG（RLU）的线性度，以LOG（效价）的关系。最后，使用刃天青或其他代谢染料细胞活力评估可以很容易地在工作流中引入，从而允许细胞毒性化合物的抗病毒帘或鉴定的化合物，导致协同杀伤与病毒12组合判断。然而，这种方法的局限性包括：荧光素酶转基因表达的病毒，这并不总是可能的要求，并具有足够允许细胞系的可用性。然而，这很可能能够适应该方法与其它报告基因的使用（ 例如 ，GFP）提供记者定量方法具有适当的线性度和信噪比。总体而言，所描述的高通量的方法可以进行修改，以适应许多不同的病毒，并适用于多种应用。

Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive &#8220;plaquing&#8221; cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9. 
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV&#8710;51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbeccos&#39; Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>NorthBio Inc.</td>
			<td>NBSF-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phosphate buffered saline</td>
			<td>Corning</td>
			<td style="width:136px;">21-040-CV</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEPES</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:136px;">BP310-1</td>
			<td style="width:251px;">Prepare a&#160; 1mM solution, pH 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alamarBlue&#160;</td>
			<td>AbD Serotec</td>
			<td>BUF012B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin potassium salt</td>
			<td>Biotium</td>
			<td>10101-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">96-well solid white flat bottom polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td align="right" style="width:136px;">3917</td>
			<td style="width:251px;">384-well plates can also be used for higher throughput</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy Mx</td>
			<td>BioTek</td>
			<td>SMTBL</td>
			<td>Monochromator microplate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquidator96</td>
			<td>Mettler Toledo</td>
			<td>LIQ-96-200</td>
			<td>96 tip manual pipetting system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Liquidator96 LTS Tips sterilized with filters</td>
			<td>Mettler Toledo</td>
			<td style="width:136px;">LQR-200F</td>
			<td style="width:251px;">Any sterile filtered tips compatible with pipettors of choice are appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microflo</td>
			<td>BioTek</td>
			<td>111-206-21</td>
			<td>Used to plate cells in a 96 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroskan Ascent FL</td>
			<td>Thermo Scientific</td>
			<td>5210450</td>
			<td>Microplate fluorometer</td>
		</tr>
	</tbody>
</table>

high-throughput titration of luciferase-expressing recombinant viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

描述了高通量方法的工作流程的概要示于图1，图2示出了一个典型的标准曲线ofVSVΔ51-Fluc的结果。四参数非线性回归分析所产生的积山从未知输入PFU（病毒滴度的估计），可以插。这些估计滴度被称为病毒表达单位（VEU）。 图3A示VEUs和滴度通过执行具有相同的样品10标准的空斑测定法得到的。样品源自其中的各种化学品使用，以增强复制和786-0细胞扩散VSVΔ51-Fluc的一个实验。 VEUs从标准曲线内插必须由这是基于样品的上清液的稀释因子乘以被转移到Vero细胞中（在这种情况下，稀释因子为5）。滴度和VEU之间的线性相关性， 如图3B所示</strong&gt;与R 2的0.8083和0.899（P &lt;0.0001）皮尔森的Ř得分。在图4中 ，从代谢法得到的典型的细胞的细胞毒性数据显示为与化学品在感染前用VSVΔ51-Fluc处理786-0细胞。最后， 图5示出了具有各种病毒获得的典型的标准曲线（单纯疱疹病毒（HSV），牛痘病毒，和腺相关病毒（AAV），所有表达萤火虫萤光素酶），并描述的孵育时间和冻融循环，根据需要。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> 图1中的工作流的高通量病毒滴定和细胞毒性测定用VSVΔ51-Fluc。 （一）种子2.5×10 4 Vero细胞每孔（100微升）细胞培养在37℃（B）24小时后转移25μl的标准曲线的对向Vero细胞（每盘2列），（C）转移25μl的样品进行滴定到剩余的Vero细胞。离心机板，并在37℃（D）在相等的量在体积的10％的孵化均匀，加入刃天青试剂含有原始样品板。培养板在37℃（E）的 3小时后，读取并记录荧光和评估细胞毒性（F）第5小时后，加入25微升2毫克/毫升的溶液的荧光素对Vero细胞的每个孔中。阅读发光并计算病毒表达单位。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> 图2。预期的VSVΔ51-Fluc的标准曲线。荧光素酶的表达是我asured内使用发光井和生物发光是用平均相对光单位（RLU）的五个不同点5小时后上清液转移。平均RLU作图对已知输入PFU / ml的解决非线性回归和生成希尔方程。两个复制曲线和标准误差线的平均值示相关（r 2 = 0.9993）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> 图的标准空斑测定滴定度与通过高通量方法3得到的比较。 （A）在PFU / ml的通过标准空斑测定在Vero细胞上获得与使用的高通量荧光素酶测定法滴定相同的样品计算的病毒滴度（VEU /毫升）进行比较（B）线性VEU /毫升之间关系的病毒滴度并通过标准纵获得滴度laque检测。线性回归曲线和决心（R2）的系数显示。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> 图4样品的可行性。样品活力之前，上清液转移到Vero细胞上通过评估使用市售的刃天青溶液细胞代谢活性测定。原始荧光值标准化为未处理，未感染的孔中。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> 图5。预计单纯疱疹病毒，牛痘，和AAV病毒的标准曲线。荧光素酶的表达是衡量五个不同点内使用发光良好，生物发光是用平均RELA略去光单位（RLU）：（A）HSV标准曲线，加入到Vero细胞接种24小时前在每孔中（100微升）和萤光素酶测定为2.5×10 4个细胞的密度被制成17小时后supernantant转移（R 2 = 0.9489，n = 1时）。甲希尔方程，通过求解非线性回归产生的（B）牛痘标准曲线，加入到Vero细胞上镀每孔（100微升）2.5×10 4个细胞的24小时更早的密度和在37℃下温育2.5小时C，之后被带到荧光素酶测定（R2 = 0.9892）。的线性方程，通过求解线性回归产生的（C）的AAV标准曲线，加入到人肺癌细胞（A549细胞）接种24小时前在每孔中（100微升）和萤光素酶测定为2.5×10 4个细胞的密度有人24小时后感染。甲希尔方程，通过求解非线性回归产生的。平均五个复制曲线和标准误差条示（R 2 = 0.9926）。

Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

High-throughput Titration of Luciferase-expressing Recombinant Viruses

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

high-throughput-titration-of-luciferase-expressing-recombinant-viruses

Research

JoVE Journal

Biology

12.8K 次观看.  Ottawa Hospital Research Institute. This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

视频: High-throughput Titration of Luciferase-expressing Recombinant Viruses

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

科研

生物学

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...