我们感谢在麻省总医院的DNA核心的约翰Darga制备的DNA筛查图书馆。 PerkinElmer公司的克里斯托弗Chigas为我们WALLAC 1420照度计的宝贵支持。史蒂芬Ciacco的和安捷伦的马丁Thomae规定的布拉沃机器人的支持。我们感谢罗恩·约翰逊和史蒂夫·提图斯在NIH慷慨地提供他们的双荧光素酶发光分析方案。

对于一个实验性的概述，参见图1。 1，准备报告质粒含有α-突触核蛋白启动子 α-突触核蛋白基因（ 图2）的调控元件跨度约10 kb的，从上游NACP（淀粉样肽的非A的β成分）二核苷酸重复序列1,2到2内含子3，我们在包括这个区域的一部分本报记者构造。我们发现，含有内含子2的成长需要特别程序质粒，概述如下。荧光素酶质粒，pGL4.10和pGL4.75（ 图3），可购自Promega公司，并且可以使用常规的分子生物学协议来传播。 <ol><li>使用配方在表1和表2中的引物扩增的2个组分的α-突触核蛋白的启动子在不同的PCR反应。 </li><li> VERIFY PCR产物上使用QIAquick PCR纯化试剂盒（Qiagen），洗脱在50μl的TE液（Tris-EDTA）的琼脂糖凝胶和清洁。存储洗脱0.9kb的片段于-20℃以备将来使用。 </li><li>消化的3.4kb的PCR片段的总体积，以及5微克pGL4.10的，用SacI和XhoI。治疗的载体以小牛肠碱性磷酸酶（Roche）和凝胶纯化使用的PureLink快速凝胶提取试剂盒（Invitrogen）中。 </li><li>用T4 DNA连接酶（NEB）结扎片段和载体。清洗用的PureLink PCR微试剂盒（Invitrogen）将完成的反应，洗脱在10μl的TE缓冲液中。 </li><li>变换2微升的清洗，连接反应成20微升ElectroMAX Stbl4感受态细胞（Invitrogen）中，并根据制造商的说明电穿孔。摇动在30℃培养箱中，在225 rpm离心90分钟。传播2卷上含有100毫克/毫升氨苄青霉素和孵化在30℃下2天。 </li><li>用牙签，选择接种4-6小菌落于2ml结核病（极品肉汤）。在30℃摇床的O / N。种植这些小量制备文化</li><li>从1.5ml每种小量培养使用的PureLink HiPure质粒小量提取试剂盒（Invitrogen）纯化的质粒DNA。 </li><li>消化小量制备用BamHI和electrophorese在琼脂糖凝胶上。正确的克隆应该产生2.8 kb的和4.9 kb的片段。 </li><li>从一个新鲜的LB（卢里亚肉汤）板的正确克隆重新划余下的小量培养和成长在30℃下2天。 </li><li>选择一个小菌落，接种1.5ml的结核病发酵剂培养物。摇O / N为30°C。不要让起子培养成长到饱和。 </li><li>淡化整个发酵剂每150ml预热结核病和动摇在30℃下2天。之前继续到下一个步骤中，使用1.5ml该培养物需额外小量制备和消化来确认克隆中replicati未变异<html>上。 </li><li>从使用的PureLink HiPure质粒大量制备试剂盒（Invitrogen）的余下培养纯化质粒DNA。这中间质粒的预期收益率是每150毫升培养50-150微克。 </li><li>解冻0.9 kb的片段定格在步骤1.2。重复步骤1.3至1.12克隆的0.9 kb的片段到中间质粒，消化，而不是用KpnI和SacI。结扎，变换，选择和成长为maxipreps如上。消化用BamHI应产生5.8 kb的和2.8 kb的片段。这是将被用于在屏幕的质粒。 </li></ol> 2，准备293T细胞中，记者质粒和DNA库的筛选 293T细胞首先被接种到96孔板中，并转染次日。报告质粒和文库的DNA稀释到96孔板中。我们在美国马萨诸塞州总医院（获得的转染级DNA文库板从DNA核心获得="_blank"&gt; dnacore.mgh.harvard.edu）。这个协议transfects单个库板染293T细胞2相同的板（ 图1），从而2细胞的板应接种每个库板进行筛选。 注:细胞生长介质中无酚红或抗生素，因为这些干扰荧光素酶检测和转染过程中增加毒性，分别为。 <ol><li>每个库板被屏蔽，重悬胰蛋白酶处理的293T细胞以1.5×10 5细胞/ DMEM中含有14％FBS的ml的浓度。倾入无菌容器（爱思进）。 </li><li>将水库，2透明底96孔板（Corning）中，和一箱过滤器枪头（安捷伦）在机器人上甲板。分配100μl的细胞分化为两个板的各孔中，为每孔1.5×10 4个细胞的密度。 </li><li>离心电池板在50×g离心2分钟，使用低斜坡速度，以确保平等贯穿每个孔中的细胞密度。在37°C和10％的CO 2 O / N。 </li><li>淡化萤火虫和海肾报告质粒至5纳克/微升的无血清培养基。在一个15毫升的锥形管结合稀释液中为5:3萤火虫到海肾质粒的比例（体积比）。 </li><li>移液管的组合的质粒到96孔板的各孔中，分配每个库板17微升，加2-10微升过剩，在每孔中。 </li><li>获得的转染级DNA文库板如上述稀释至13.3纳克/微升用无内毒素的水。每孔的最小体积应为28微升。 </li></ol> 3，进行转染在屏幕的第2天，报告质粒和文库的DNA转染到293T细胞中。 <ol><li>对于每个库板，以聚苯乙烯试管混匀107.5微升RT Optifect（Invitrogen公司）的4.3毫升血清的培养基（1:40稀释）。孵育5分钟，在室温和Ñ​​免除44微升（每个库板）到96孔聚苯乙烯V形底平板（Greiner）中的每个孔中。 </li><li>放置板的稀释Optifect，报告质粒（步骤2.5），库的DNA（步骤2.6），和一个盒上的机器人甲板枪头。 </li><li>吸17微升报告质粒，43微升稀释Optifect，和26微升的DNA库中，每次插入抽吸之间的5微升的空气间隙。该文库DNA应该最后吸气，以防止交叉污染。免除提示的内容到一个新的聚苯乙烯V形底板，混合，盖，并预留20分钟，使脂质/ DNA复合物的形成。如果转多个库板，更换枪头和库板，并重复。 </li><li>揭开Optifect / DNA混合物板，并将其放置在机器人上甲板2板293T细胞。使用单一框的枪头，吸80微升的Optifect的:DNA混合物，慢慢地免除40微升到细胞的每块板。如果反fecting多个库板，重复所有Optifect:DNA的板块。覆盖细胞。 </li><li>离心细胞板在1200×g离心30分钟。覆盖，并返回到培养箱中。 </li><li>于室温孵育4-6小时。在此培养后，通过混合12毫升DMEM含10％FBS和10微克/毫升环丙沙星（的CellGro）对于每个转染的库板制备的新鲜培养基。倒入新鲜介质装入无菌水库。 </li><li>将2盒机器人的提示，细胞的单盘，含有新鲜媒体和废物储存到机器人甲板上的水库。从细胞中吸出100μl的媒介，分装到废物储存器部分地填充有70％的乙醇。使用新鲜的秘诀，吸100微升新鲜培养基，慢慢地分配到细胞。重复单元的各个板块。 </li><li>返回板的培养箱中培养48小时。 </li></ol> 4，进行双荧光素酶检测在屏幕的第4天，在萤火虫和海肾萤光素酶测定法进行。 注:双萤光素酶测定，需要顺序加入2测定缓冲液，3X萤火虫测定缓冲液和3X Renilla荧光测定缓冲液中，如表3中描述的萤火虫和海肾萤光素酶不分泌，从而细胞必须测定荧光素酶活性之前裂解。 。萤火虫测定缓冲液中裂解细胞，并提供了底物的萤火虫荧光素酶; Renilla荧光测定缓冲液淬灭萤火虫信号且提供基片的海肾荧光素酶。 <ol><li>每个库板，如表3中所述制备8毫升的3X萤火虫测定缓冲液从储液，并免除82微升到96孔V形底的板的各孔中。 </li><li>每个库板，还准备12 3X Renilla荧光测定缓冲液中，并分配122微升到96孔V形底的板的各孔中。抛开两者的萤火虫和海肾检测缓冲区。 </li><li>放置一个方块的枪头，一个废物储存器，和2板的细胞（来自相同的库板转染）在机器人上甲板。 </li><li>从各板吸60微升媒体并丢弃在废物储存部分地填充有70％的乙醇。盖板及待用。如果转多个库板，重复板的所有集合。 </li><li>放置2盒枪头，3X的萤火虫测定缓冲液中的板，并在机器人上甲板的一组细胞的板。 </li><li>吸出40微升3X萤火虫测定缓冲液中，并将其添加到细胞中的所述第一板，充分混合。切换至新的枪头，然后为第二单元板。把细胞在室温下10分钟，完成裂解。如果转多个库板，更换的提示和电池板的框，然后重复。 </li><li>从每个细胞板在一个光度计，记录为1秒，每孔的每个孔中记录的发光。返回板到机器人甲板。 </li><li>放置2盒枪头，3X的Renilla荧光测定缓冲液中的板，并在机器人上甲板的一组细胞的板。 </li><li>吸出60微升3X Renilla荧光测定缓冲液中，并将其添加到细胞中的所述第一板，充分混合。切换到枪头一个新的框，然后为第二单元板。如果转多个库板，重复单元板的所有集合。 </li><li>从光度计所有板块记录发光，记录每孔1秒如上。丢弃板。 </li></ol> 5。数据分析<ol><li>计算萤火虫: 海肾荧光比（"F:R比率"）由海肾萤光信号的计数除以萤火虫信号的计数每口井。 </li><li>通过将在F计算感应值:每个孔的平均F R的比值:所述板的R比，不包括孔中的最高和最低的25％。 </li><li>跨重复平均感应值为一个单一的转库板。基因诱导或抑制α-突触核蛋白比三褶皱更被认为是"点击"在此屏幕上，并受到进一步的验证和二次筛选。 </li></ol>

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这项工作是由发牌BacMamreagents（美国专利5731182）获得特许权使用费的支持。

α-突触核蛋白有牵连的帕金森氏病（PD）作为路易体4，考虑特殊病征为疾病细胞内的夹杂物的成分。众多的全基因组关联研究已发现，单核苷酸多态性在α- ​​突触核蛋白与风险偶发性帕金森氏5,6,7增加。虽然比散发性PD不太常见，家族性帕金森病也可在α- ​​突触核蛋白8所造成的突变，以及复制和α-突触核蛋白基因9,10,11的三重，这种现象也出现在散发性PD ...

找出关键基因的调控元件，并在他们身上起作用的因素的能力是根本的为众多生物过程的探索。然而，识别，调节基因表达的稀有细胞类型的因素，如特定神经元群体，是具有挑战性的。在这里，我们提出了一个协议，用于识别α-突触核蛋白（SNCA）的新的转录调节，与帕金森氏症有关，表现在多巴胺能神经元在黑质的基因收杆中脑的致密区。我们使用的是高通量，双荧光素酶， 在体外记者屏幕解构α-突触核蛋白表达在293T细胞中实现这一点。 α-突触核蛋白的启动子是第一个克隆到含有萤火虫荧光素酶基因的报告质粒。含有组成型活性的启动子的控制下的海肾荧光素酶的市售质粒作为内部对照。成分股ë记者构建体共转染到293T细胞中微孔板与从DNA表达文库质粒，使得每孔转染了一个单一的文库质粒。 48小时后，对于每一个记者荧光素酶活性被依次使用双辉光测定法测量。在每个孔中的海肾荧光素酶的活性（F:R比率）板基于归一化后的α-突触核蛋白的响应于每个文库的基因的相对表达通过的萤火虫的比率推断。 该协议提供了筛选大量的基因（〜2,500每星期）就其使用最少的人力（1-2人），最低的成本（约300试剂费用为每微孔板检测），以式激活报告基因的能力的能力。神经细胞的基因的转录调节子（ 如 ，α-突触核蛋白），这是很难培养的神经元耐火遗传操作来研究，可以比在此直接功能测定并排侧。我们在包括我们协议的详细方法进行克隆和含有α-突触核蛋白的启动子的质粒的增长，因为我们发现，当用常规方法（见讨论）生长含有该区域的质粒是不稳定的。我们还包括了一种廉价的替代市售的双荧光素酶试剂，用于高通量测定法。这个协议可以很容易地适应目标的感兴趣的其它调控元件，或以任何需要高通量的瞬时转染过程。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>QIAQuick PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K2100-12</td><td></td></tr><tr><td>PureLink PCR Micro Kit</td><td>Invitrogen</td><td>K310250</td><td></td></tr><tr><td>PureLinkHiPure Plasmid Miniprep Kit</td><td>Invitrogen</td><td>K2100-03</td><td></td></tr><tr><td>PureLinkHiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K2100-07</td><td></td></tr><tr><td>Bravo Robot</td><td>Agilent</td><td>-</td><td></td></tr><tr><td>Robot Pipet Tips with Filter</td><td>Agilent</td><td>19477-022</td><td></td></tr><tr><td>Robot Pipet Tips without Filter</td><td>Agilent</td><td>19477-002</td><td></td></tr><tr><td>Clear-bottomed 96-well Plates</td><td>Corning</td><td>3610</td><td></td></tr><tr><td>Reservoirs</td><td>Axygen</td><td>RES-SW96-HP-SI</td><td></td></tr><tr><td>Polystyrene V-bottomed 96-well Plate</td><td>Greiner</td><td>651101</td><td></td></tr><tr><td>Polypropylene V-bottomed 96-well Plate</td><td>Greiner</td><td>651201</td><td></td></tr><tr><td>Adhesive Plate Covers</td><td>CryoStuff</td><td>#FS100</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Phusion DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>BAC 2002-D6</td><td>Invitrogen</td><td>2002-D6</td><td></td></tr><tr><td>Calf Intestine Alkaline Phosphatase</td><td>Roche</td><td>10713023001</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>pGL4.74</td><td>Promega</td><td>E6931</td><td></td></tr><tr><td>ElectroMAX Stbl4 Competent Cells</td><td>Invitrogen</td><td>11635-018</td><td></td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>Invitrogen</td><td>18265-017</td><td></td></tr><tr><td>DMEM without Phenol Red</td><td>Invitrogen</td><td>31053-028</td><td></td></tr><tr><td>Optifect</td><td>Invitrogen</td><td>12579017</td><td></td></tr><tr><td>Ciprofloxacin</td><td>CellGro</td><td>61-277-RF</td><td></td></tr><tr><td>Tris-Hydrochloride Powder</td><td>Sigma</td><td>93287</td><td></td></tr><tr><td>Tris-Base Powder </td><td>Sigma</td><td>93286</td><td></td></tr><tr><td>Triton X-100 Pure Liquid</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>DTT</td><td>Invitrogen</td><td>15508-013</td><td></td></tr><tr><td>C&amp;#8211;nzyme A</td><td>Nanolight</td><td>309</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A6419</td><td></td></tr><tr><td>Luciferin</td><td>Invitrogen</td><td>L2912</td><td></td></tr><tr><td>h-CTZ</td><td>Nanolight</td><td>301</td><td></td></tr><tr><td>PTC124</td><td>SelleckBiochemicals</td><td>S6003</td><td></td></tr></tbody></table>

high-throughput functional screening using a homemade dual-glow luciferase assay

我们提出了一种快速，廉价的高通量筛选的协议，以确定α-突触核蛋白与帕金森病相关的基因的转录调节。 293T细胞瞬时转染从一个排列的ORF表达文库质粒连同荧光素酶报告质粒，在1个基因的每孔微孔板格式。 48小时，以确定每个基因库后的α-突触核蛋白的转录，归表达从内部控制结构（一hCMV启动子指导海肾萤光素酶）的影响后，萤火虫荧光素酶活性测定。该协议是由封闭在一个生物安全柜台式机器人，它在96孔板进行无菌液体处理提供便利。我们的自动转染协议很容易适应高通量慢病毒库生产或需要三转染的大量独特的文库质粒的共轭等功能筛查方案nction用一套共同辅助质粒。我们还提出一种廉价的和验证的替代市售的双荧光素酶试剂，它采用PTC124，EDTA和焦磷酸盐来抑制萤火虫荧光素酶活性之前， 海肾萤光素酶的测定。使用这些方法，我们筛选7,670人的基因并确定了68监管α-突触核蛋白。这个协议是很容易修改的目标感兴趣的其他基因。

典型的荧光素酶值，F:R比和为一个单一的半板的感应值被显示在图4中注井H2，32倍诱导的命中。基因会引起过度的毒性（例如，井E3），或井转染较差，会产生低的值既萤火虫和海肾萤光素酶，但平均感应值。基因会引起非特异性诱导荧光素酶活性，也许是通过与pGL4骨干相互作用，将导致萤火虫和海肾萤光素酶一样，产生的平均感应值。大的差异在重复之间的感应...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

我们目前确定采用高通量机器人转染和一个自制的双发光荧光素酶检测转录调控因子的快速和廉价的检查方法。该协议迅速产生直接并排端功能数据数以千计的基因，很容易修改针对任何感兴趣的基因。

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

high-throughput-functional-screening-using-homemade-dual-glow

Research

JoVE Journal

Biology

23.4K 次观看.  Massachusetts General Hospital. 我们目前确定采用高通量机器人转染和一个自制的双发光荧光素酶检测转录调控因子的快速和廉价的检查方法。该协议迅速产生直接并排端功能数据数以千计的基因，很容易修改针对任何感兴趣的基因。 

视频: High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

癌症研究

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

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 ...

免疫与感染

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 ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

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.

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 ...

ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids

Source: Higa, A. et al. <a href="https://app.jove.com/v/62668">High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, single-addition assay to assess cell viability in patient-derived organoids upon treatment with a test agent using intracellular ATP levels as a marker for cellular metabolic activity. The intracellular ATP levels are measured using the luciferase/luciferin reaction, where the emitted light intensity indicates the cellular viability and, in turn, the test agent&#39;s growth-inhibitory potency.

基于 ATP 的荧光素酶活力测定:一种评估测试剂对肿瘤类器官生长抑制潜力的均相方法

Source: Higa, A. et al. High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids. J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

To evaluate the luminescent signal from the bioreporter using a luciferase assay, first, prepare the reaction components, and use the supernatant as the source of the bioreporter.
Dilute the LgBiT and substrate to 1x before use. Transfer 50 microliters of the supernatant from each well or tube, to a 96-well plate, and add 50 microliters of 1x LgBiT to each well. Incubate for 5 minutes at room temperature.
Open the luminometer software. Add 50 microliters of 1x substrate to each well. Place the plate in the luminometer and run the software.