We thank Eliq Oster for the images of the Bacillus subtillis. The work is supported by the European Research Council (# 260871) and the Israel Science Foundation (#592/10).

（一）建设安装<ol><li>选择一个平板扫描仪光学分辨率：4,800 dpi的;硬件分辨率：高达4,800 x 9,600 dpi的;色位深度：48位，可以在相关的温度和湿度水平进行工作。 </li><li>培养皿有显著体积，不像纸通常用于商业扫描器。菌落趴在琼脂层的顶部，约5毫米以上的扫描仪的表面。注：对于大多数扫描仪的殖民地将成为市场关注焦点。 </li><li>有些扫描仪公司不允许同一类型到同一台计算机的扫描仪的多个实例的连接。确保一个以上的扫描仪可以连接并确定了独特的。 </li><li>准备详情如下配件： <ol><li>无菌黑色的制备条毡布覆盖板（ 图1中的步骤2）。 </li><li>要保持培养皿到位，准备白色板支架（材料和方法）适合的扫描仪和具有6 HOLES菜肴的大小（ 图1步骤3）。 </li></ol></li><li>如果需要的微生物的生长，将扫描仪在温度受控的环境中。连接扫描仪到计算机。 </li><li>可选：继电器连接到计算机并绕过扫描器的通/断开关，以克服温度梯度。 注：该继电器单元是极其温度敏感的微生物。它关掉扫描仪，使电子不热的表面不均匀。使用扫描仪表面只有一部分可以达到类似的结果。 </li></ol> 2，安装扫描管理器<ol><li>从http://www.phys.huji.ac.il/bio_physics/nathalie/publications.html所有“扫描管理器”应用程序文件复制到指定的目录中。 </li><li>根据扫描仪的名称配置ScanningManager.exe.config文件，因为它们出现在计算机管理 - &gt;设备管理器 - &gt;成像设备（ <stro纳克&gt;图2）。 </li><li>对于“扫描管理器”应用程序的故障排除，请参阅自述文件。 </li><li>有关“扫描管理器”应用程序进一步的文件，见Documentation.html。 </li></ol> 3，进行实验<ol><li>准备陪替氏培养皿（ 图1）： <ol><li>淡化文化约2,000 CFU /毫升。板均匀分布在板面上0.1毫升文化。 </li><li>为了获得殖民地和菜之间良好的对比度并吸收水分，覆盖板用一块无菌黑毡布。把盖子上盘。 </li></ol></li><li>将板在扫描仪的持有人。 </li><li>启动扫描管理器（ 图3）：从连接的扫描仪列表中的参与扫描器。选择将要采取（重复次数）的图像的数量，随后的图像之间的时间间隔（时间间隔），并起动吨前的延迟时间他试验（开始）。 </li></ol>该图像4。分析注：提供用于分析在MATLAB图像使用“ScanLag”，这是免费提供给非商业用途的http://www.phys.huji.ac.il/bio_physics/nathalie/publications的计算方法的概要。 HTML。提供更多功能详见软件说明书，并通过实例代码补充。 <ol><li>从每个扫描仪的图像分类到不同的文件夹。 </li><li>利用Matlab的命令窗口中运行以下程序。运行这些程序所需的参数在软件手册中规定。 <ol><li>运行PreparePictures进行预处理：在预处理，从扫描仪的图像对准。每个培养皿从图像中每个序列裁剪。在其中的每个图像被收集的时间是从时间标记检索。 </li><li>运行TLAllPlates进行检测和跟踪在电子ACH培养皿分开，菌落检测并分配一个识别号码。 </li><li>所提取的数据的分析：每个检测到的菌落的大小测量为时间的函数。要提取菌落的外观分配使用Matlab的命令窗口中执行以下功能（在软件手册相关的信息是可用的）： <ol><li>运行ScanLagApp与菌落的曲线图一起显示某一培养皿的分析大小与时间的关系（ 图4）。 <ol><li>使用滑块来改变极板的当前时间。的时序也将在菌落面积图形匹配的垂直线表示。 </li><li>点击一个殖民地，看看它的相关曲线，反之亦然，单击曲线上找出其关联的殖民地。识别所述菌落后，其表型可以通过从所识别的培养皿中挑选菌落进行检索。 </li><li>在自动分析了噗过滤器缺陷唱了殖民地，并单击排除按钮。当点击排除菌落数将变为黄色。 </li></ol></li><li>经历了各板块的分析结果，总结的结果： <ol><li>运行AddHistograms打造的外观倍的直方图。 </li><li>运行plotDeathCurve创建分布的生存曲线表示。 </li><li>运行GetAppearanceTimes让所有殖民地出现的时间在实验中。 </li></ol></li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

基于直接观察显微方法通常被认为是“黄金标准”研究单细胞行为。 ScanLag，这使的滞后时间中的细菌种群分布的测量，提供了数据与从单细胞分析中得到的分布基本一致，并实现更高的统计信息。 几个关键步骤必须对这种方法进行很好地工作：第一，不要在扫描仪的分辨率，这应该是高达4,800 x 9,600，以获得良好的图像妥协。其次，要知道，没有电源管理模块（步骤1.6），温度梯度可能发展放在平板表面，影响成长。另一个重要步骤是可能已经被软件被误计为菌落诸如灰尘的缺陷的过滤。该软件的内置背景减法通常克服了这些缺陷，但有时是“假”的菌落有手动过滤。 主要的故障排除步骤可能会解决板间变异的设置，由于以下几个原因：（一）微生物的生长速度受温度的影响。不同的温度和湿度条件可能是由于在孵化器的菜通风不均或位置。 （b）该扫描仪的电子设备可能会发热，导致在整个扫描的表面上的温度梯度， 图10示出了在芽孢杆菌属细菌，例如空间的热梯度的影响下，连同之前放置在扫描仪的表面温度的测量值和实施电源管理模块后（步骤1.6）。注意，该模块可能没有必要为较新的扫描器，并且如果在扫描仪面的一部分的情况下，电源管理可能是不必要的。 （c）板材具有不同的不透明度琼脂营养可能导致不同的检测级别。 图11显示了日Ë容忍不同体积的营养琼脂导致不同的混浊。 实验的ScanLag技术是容易执行，只需要直接制版标准的培养皿的微生物，并且将它们放置在扫描仪的表面。被开发了的软件控制整个过程。图像采集是自动完成的，并且图像分析用于菌落跟踪也是自动进行的。此外，该系统可以按比例增加来测量许多不同的条件在同一时间。最后，该方法依赖于商业办公扫描仪，因此，成本低。 该技术可以扩展到微生物的从大肠杆菌不同的研究大肠杆菌 K-12，但几个因素必须作出。首先，早期出现的菌落在后来的影响必须被评估，因为在以前的出版物7进行详细描述。为E.大肠杆菌 K-12，本每盘CFU的最大密度为200，其他菌株不同大​​小的菌落，和殖民地之间的其他可能的串扰，可能需要不同的密度。第二，在板的分析校准到可能需要进行修改，这取决于固体介质和集落之间的对比是基于特定软件的阈值。该软件可以扩展还提取其他菌落特征，超越滞后和增长速度。软件代码是开放的，可以进行修改，以提取菌落的形状，亮度，平滑度和颜色。 因为测量上的菌落，从单个细胞起源制成，该数据表明，不能用群体水平的测量被测量的新的表型。滞后时间分布的重要性是显示在抗生素存在下进行。许多抗生素被称为是有效的只有对生长的细胞;因此只要细胞停留在滞后期，不长，他们是亲从这些抗生素21效果tected。当幸存细菌的数量都在时间间隔内的抗生素治疗15,22,23评价，亚群的细菌，称为持久化，常常显露。在某些情况下，免疫力抗生素治疗源于长期滞后。利用此设置，这种滞后显露并经常具有一个非平凡分布24（ 图6）。这个方法还可以使稀有突变的检索。例如，电镀诱变的种群后，突变体可以基于表型鉴定，并直接分离，而不选择。该设置还可以监测细胞间的相互作用，通过测量菌落生长由于附近的菌落密度的函数和量化，如群体感应或蜂拥细菌的传播现象。通过使用这种方法今后的实验中发现了多维度的信息会导致微生物种群更好地表征s和在医学和环境研究的进展。

细菌已经进化到了许多紧张的情况作出反应。适应压力的一个普遍特征是增长动力1,2的变化。指数增长率与滞后时间，即要适应新的情况所需要的时间：增长动力由两个参数大多特点。而大量的高通量方法侧重于增长速度的测量，滞后时间往往是一个被忽略的参数，但它是为适应新形势的特性的关键参数。在传统上一直使用费力的手工方法3,4进行适应的定量变化的条件。最近，先进的技术已经开发了用于单细胞5,6的分析。然而，它仍然是难以区分正常生长下列中生长延迟（即延迟时间）2,3从缓慢增长。一个自动化的方法构建，ScanLag 7，这两个相似的表型之间的区别。 滞后时间分布的研究的重要性一个突出的例子是在抗生素治疗透露。许多抗生素和其它应力仅杀死活跃生长的细胞，并且，因此，细胞被“滞后”被保护的8。为了监测滞后时间，人们可以观察单个细胞在显微镜下和监测时间第一师。这种方法的一个主要缺点是，动态时间范围是有限的;那些早期生长的那些细胞覆盖表面以指数速率，有效地降低细胞的生长具有较长的滞后时间。使用的流动室有所规避这个5，但细胞数目有限的可跟踪和细菌，退出延迟期后的大多数人口已开始生长的小部分不能被观察到。尽管有这些限制，一些研究已经评估二叔水平的影响采用单细胞显微9-12上的滞后时间分布fferent强调。另一种方法来监视滞后时间分布是通过浊度测量13,14。许多平行的培养物，从单个小区中的每个开始，生长在其测量细菌的培养液中的数目随时间的光密度读取器。此方法是由外推法的精度和孔中的板的数量的限制。 一个自动化的方法被开发，被称为ScanLag，使滞后时间和生长时间分布进行评估，即使是细菌的小部分的人口有很长的滞后时间。该方法是基于检测的常规营养琼脂平板15（ 图1）的菌落。若要自动检测16,17商用扫描仪18是由内部软件定向定期收购板图像阵列，被设计。软件设计为自动分析图像，并提取量化参数19,20，如外观各菌落，其在这里被定义为时间从20个像素长到80个像素的每一个集落生长和时间的时间。这里，我们提出的方法的细节，包括该系统的建立和使用已被改进以提供更好的用户界面的自动图像分析软件。 该方法可用于测量其他特征的菌落，如形态和颜色。这些类型的参数的测量将允许在多维表型组学系统的使用。作为该方法检测来自单个细胞的菌落，该系统可以显示，可以不通过群体水平的测量被测量的新的表型。该技术使罕见的变异的检测和检索，并有利于筛选所需要的特性。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Material</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:71px;">BD</td>
			<td align="right" style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Difco LB broth</td>
			<td style="width:71px;">BD</td>
			<td align="right" style="width:119px;">240230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish 90mm</td>
			<td style="width:71px;">Miniplast</td>
			<td style="width:119px;">20090-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Epson Perfection v37</td>
			<td style="width:71px;">Epson</td>
			<td style="width:119px;">B11B207201</td>
			<td style="width:167px;">Flatbed Scanner with Optical resolution: 4800 dpi, Hardware Resolution:&#160;4800 x 9600 dpi, color bit depth 48-bit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epson Perfection v200</td>
			<td style="width:71px;">Epson</td>
			<td style="width:119px;">B11B188011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epson Perfection 3490</td>
			<td style="width:71px;">Epson</td>
			<td style="width:119px;">B11B177011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ADU200 USB Relay</td>
			<td style="width:71px;">ONTRAK</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">pieces of sterile black felt cloth</td>
			<td style="width:71px;">custon made</td>
			<td style="width:119px;"></td>
			<td>approximatly 100 x100 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">white plate holders</td>
			<td style="width:71px;">custon made</td>
			<td style="width:119px;"></td>
			<td>surface size, with 6 x 99mm diameter hole</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Delerin rings the size of the Petri dish</td>
			<td style="width:71px;">custon made</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">inner diameter: 72 mm 
			outer diameter: 86 mm 
			top outer diameter: 90 mm</td>
		</tr>
	</tbody>
</table>

scanlag: high-throughput quantification of colony growth and lag time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

作为延迟时间的分布提取一个例子， 图5示出了识别和跟踪每个集落具有在一个板上的具体特征随着时间的推移，由于将在筛选测定来完成的ScanLag的能力。 当微生物的培养是异构的，不同的亚群可能在试验过程中显露出来。例如， 图6示出的外观量化，从而揭示了双峰滞后时间分布在大肠杆菌的突变株大肠杆菌。 细胞的生长速度影响菌落的出现时间。当菌落生长以同样的速度，出现较晚可以归因于滞后时间（ 图7）。 为了验证该方法确实测量单个细胞的滞后时间，我们比较ScanLag结果与使用单细胞显微镜获得的;这种验证是递减在以前的出版物7 ribed的细节。此方法使监测更多的细胞比用显微镜进行评估。使用显微镜和使用ScanLag取得所得的分布在很大程度上重叠。该ScanLag分布稍宽;理论分析预测扩大为划分时间的标准偏差的顺序。如果生长时间是不同的每个菌株，在外观上与延迟的来源必须进一步用其它方法影响。 的早期出现菌落后菌落外观的影响进行了研究。对照实验7确认后的外观没有影响，只要每个平板菌落的总数没有超过200（ 图8）。另一种控制实验证实，殖民地上盘的位置没有影响其出现的时间7。 的p的分析鲈被校准以取决于存在于生长培养基中或微生物的类型的营养物质，可能需要修改特定的阈值。然而，该分析是相当健壮的大范围的阈值， 如图所示9。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig1highres.jpg" width="500" /> 图1：创建一个殖民地的外观分布所需的步骤示意图。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig2highres.jpg" width="500" /> 图2的屏幕抓图设备管理器中显示的扫描仪附带的配置文件，以及姓名。 <a href="https://www.jove.com/files/ftp_upload/5456/51456fig2highres.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig3highres.jpg" width="500" /> 图3的屏幕抓图扫描经理。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig4highres.jpg" width="500" /> 图4，屏幕截图ScanLagApp的。板的图像是在左边窗格中，每个菌落的生长曲线在右窗格中，如手册中详细说明。当两个或多个菌落合并在一些菌落面积曲线的尖峰出现。当殖民地合并发生时，这些殖民地的面积被认为是联合区。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig5highres.jpg" width="500" /> 图5。1板的分析的代表性结果：（A）的检测应用在实验结束时，输出的图像。每一集落区域被识别，彩色和分配有一个唯一的ID号。箭头指向B.测量基于强度阈值检测菌落（代表菌落在LB琼脂或M9琼脂大肠杆菌菌落这个阈值是0.03，其他媒体或细菌，这种阈值可以在函数ProcessPictures调整）。上述10个像素只有对象计数（此阈值可以在函数MatchColonies进行修改）。检测殖民地开始于大约10 5细菌。 （B）图解中的像素与在该板4代表菌落时间的区域。各殖民地的“演出时间”是在检测到殖民地时。每个菌落的&#39;成长时间“在这里定义为在时间来回成长米20个像素至80像素（这些边界是可调节的使用功能getAppearanceGrowthByVec）。该软件不包括在达到上限之前合并的殖民地。特定菌落根据其特征识别是容易由于该识别号码和分配给每个集落的颜色。例如菌落＃1，56，77和124的两个图形突出。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig6highres.jpg" width="500" /> 图6。外观量化可以发现双峰滞后时间分布的出现的次数为两个不同的菌株ScanLag分析直方图的比较。蓝线：野生型​​菌株成倍增长（总计：1,320殖民地）;红线 - 高持久性突变富含落后菌（共1,529殖民地）。 （ 一 ）归一化的外观直方图。高峰外观呈指数生长的细胞中是典型的时间长到可检测的菌落。插图：减去指数文化高峰的时间来获得实际的滞后时间对数间隔箱显示双峰滞后时间分配后的突变株相同的直方图。 （ 二）对数刻度的相同的数据（A）的生存功能。这种表示增强了突变株的后期外观尾巴。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig7highres.jpg" width="500" /> 图7。外观分布和生长时间的分布（A）和（B）表示的外观时二维直方图和在两种不同的条件（该软件不包括菌落到达上限之前合并）的生长时间。 （c）比较直方图两个菌株的显示出现的时间差异，而生长时间（D）是相似的。 <a href="https://www.jove.com/files/ftp_upload/51456/51456fig7highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig8highres.jpg" width="500" /> 图8。早期殖民地的外观不会干扰以后的菌落的外观。（一 ）单独滞后镀野生型细胞的时间分布。 （二）单独滞后感冒敏感菌株的时间分布，转移到允许的温度后。 （C） 的滞后相同的条件下电镀一起两种菌株的时间分布。黑线表示基于分别测量各应变时所获得的数据的预期分布。一个好一个只要每个平板菌落的总数是200以下，获得预期的和测量的分布之间greement。 <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig9highres.jpg" width="500" /> 。图9中的检测阈值不影响菌落外观分布的形状的菌落外观分布为使用两个不同的阈值的大小相同的数据集进行分析; （ 一 ）阈值大小为10像素; （B）阈值大小为50像素。每个直方图细胞数：约。 1500元。不同的检测阈值的效果只有在检测时间的转变。 <img alt="图10" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig10highres.jpg" width="500" /> 图10。温度稳定性的测量ACR一盘用生长在无电源管理扫描器枯草芽孢杆菌菌落OSS扫描仪的表面。（A）图片。表面上的底变暖，因此，菌落生长得更快。板旁边的渐进线是热梯度的示意性表示。 （B）的温度稳定性的测定使用两个热电偶放置在扫描仪的表面。扫描仪在每次扫描过程中，并随时间不断升温。 （c）与电源管理，温度均匀，菌落生长在类似的速度。 （ 四）同（B）与电源管理模块。为±0.2°C的温度稳定性<img alt="图11" fo:content-width="5in" src="/files/ftp_upload/51456/51456fig11highres.jpg" width="500" /> 图11。在外观上分布的差异，可能是由于二fferences在固体培养基的体积。相同的细菌的培养物铺于不同体积的LB琼脂，造成琼脂​​表面的不同高度。的不同高度导致了不同的不透明度的板，因此，不同的检测时间。间板差异被检查和高度不同条件之间的差异。 30±0.5毫升LB培养基琼脂等体积的板之间（A）典型的差异。不同高度条件之间（B）差异：外观分布进行了测定和40±0.5毫升（蓝色）30±0.5毫升（绿色）用于板与20±0.5毫升（红色）卷。每个条件是6个不同的板的平均值。只要板的体积是在±5毫升不等，不透明度是相似的，导致类似的外观的时间分布。

Watch this Scientific Journal Video about ScanLag: High-throughput Quantification of Colony Growth and Lag Time at JoVE.com

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

ScanLag：高通量的菌落生长定量和滞后时间问题

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

scanlag-high-throughput-quantification-of-colony-growth-and-lag-time

Research

JoVE Journal

Immunology and Infection

16.0K 次观看.  The Hebrew University of Jerusalem. ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

视频: ScanLag: High-throughput Quantification of Colony Growth and Lag Time

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, ...

科研

免疫与感染

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...