我们承认资助CPRIT（RP100119）和韦尔奇基金会（I-1665）。

整个协议约需4天。 1。制备的siRNA和记者转染的细胞我们将在这里介绍使用一小部分的siRNA（384不同的siRNA池阵列每个池组成的4倍siRNA靶向单个基因在96孔板格式）从全基因组siRNA文库，仅供询问的siRNA库的协议。对于这个特定的研究中，我们将利用HCT116细胞表现出离经叛道的Wnt和KRAS信号，p53的活性。合适的细胞系的研究，旨在询问其他细胞的过程，还必须以类似的方式被识别和评估。 <ol><li>洗涤5×10厘米2块板的80-90％汇合的HCT116细胞用PBS然后收获细胞，用胰蛋白酶消化，用1毫升的胰蛋白酶消化溶液/板，接着用10ml的Dulbecco氏改良的Eagle培养基（DMEM）中，含2％铁河谷牛血清（FBS）和1％青霉素/链霉素/板。 </li><li>将悬浮的细胞转移到50毫升锥形管，离心〜130×g离心5分钟，收集上清，重悬细胞沉淀在10毫升的DMEM培养基/ 2％FBS / 1％青霉素/链霉素。 </li><li>与细胞计数器计数细胞数，然后使一个细胞10 5细胞/ ml的溶液，保持150毫升细胞悬浮液中的下一个步骤。板10 4个细胞（100微升）在使用多点自动液体分配器（以下简称多点）在96孔细胞培养板的各孔中。在这种情况下，电镀12×96孔板中，应足以将细胞悬浮液。 </li><li>孵育板与细胞在37℃培养箱中，5％的CO 2，同时准备转染混合物。 </li></ol> 2。准备记者构造联合解决方案<ol><li>隔离高品质的记者使用:标准midiprep套件（NucleoBond的Xtra南部生产的构建DNAClontech）中，并稀释至终浓度为1毫克/毫升的每个记者的DNA。 </li><li>准备100μl的记者构造相结合的p53-FL，30微升30微升8X TCF-RL，30微升Gal4的ELK1，6微升UAS-CL和4微升H 2 O来实现的DNA混合储备溶液最后的记者1:1:1:0.2比例。该DNA混合原液DNA终浓度为0.3毫克/毫升。 </li></ol> 3。准备的siRNA / DNA转染混合物在这个协议的一部分，我们会做好准备，这将是足够的转染12×96孔板的siRNA / DNA转染混合物。 4×96孔板的siRNA库对于这个测试，我们将在一式三份的siRNA转染每个池。转染试剂，我们将使用被称为Effectene（Qiagen公司）。在我们手中，这是唯一的试剂适用于siRNA和DNA导入培养细胞的同时交付。 <ol><li>记者构造股票溶胶稀释80微升ution在8ml的EC缓冲液（Effectene套件）中，实现了与终浓度为3微克/毫升的DNA溶液。在一般情况下，准备足够的DNA溶液混合，可直接使用。 </li><li>板20微升该DNA溶液在96孔/板的各孔中。 96孔板的数量将取决于有多少siRNA的池将被测试。例如，在这种情况下，我们将需要4×96孔板评估384座不同的siRNA。 </li><li>要测试的siRNAs的，应该是股票的浓度为5μM。如果没有，也可以是镀副本和与每个库的使用推荐的稀释缓冲液稀释至浓度。 </li><li>将2微升每5微米的siRNA股票到相应的PCR板稀释的DNA与Biomek自动化液体处理股票。根据不同的研究规模，多通道移液器可能就足够了。 </li><li>现在，我们将增加1微升增强溶液（套件Effectene），以每口井的DNA / siRNA的解放军德。这又可以实现与自动化液体处理多通道移液器。 </li><li>允许的增强反应发生在室温下5分钟，然后加入3微升Effectene转染试剂的各孔中，并在室温下孵育10分钟。 </li><li>要停止的转染复合物的形成，PCR板的各孔中的DMEM加10微升/ 2％FBS / 1％青霉素/链霉素。 </li><li> 10微升转染混合物转移到含有细胞（从细胞培养箱中检索）的96孔板的各孔中。由于每个转染将一式三份进行，相同的组合将被应用到两个额外的96孔板中的细胞。 </li><li>孵育的细胞的转染混合物在37℃下加入在湿润的环境中，5％CO 2的36小时。 </li></ol> 4。确定细胞样本的荧光素酶的活动 36小时后，准备测量荧光素酶活性在转染细胞。端点应在每个实验的基础上确定。在我们的研究中，培养时间36小时以前产生了足够强大的信号，这意味着结果的决心。由于本研究中采用多个记者（1分泌到培养基中，另外两个细胞的细胞质中表达），我们将获得从两个培养基以及细胞裂解液中的测量。 <ol><li> CL在培养基中的活性检测: <ol><li> 20μl的培养基是副本镀在一个白色的不透明的96孔板中（无论是使用Biomek的液体处理器或者多通道移液器）。 </li><li> CL测定缓冲液中加入20μl（定位系统）加入到各孔中。 </li><li>向每孔加入10μl的海萤荧光素底物（定位系统），并使用光度计（由BMG Pherastar板读数器例如）检测CL活动。 </li></ol></li><li> FL和RL活性检测: <ol><li>裂解的c通过首先除去培养基中的细胞l。这可以通过转动板倒置和扔在一个接收器中的介质中迅速完成。剩余介质可以去除纸巾轻轻拍打倒挂板。加入30微升的1X被动裂解缓冲液（Promega公司），每口井的细胞，使用多点的地方在一个平台上摇臂（BELLCO是一个公司，使这些）设置一个中等的速度为5分钟，在RT。 </li><li>加入20微升的萤光素酶检测试剂II（部分Promega公司的双荧光素酶试剂盒）使用光度计使用多点，并立即测量FL活动。接下来，添加停止格洛试剂（Promega公司），将淬火佛罗里达州的活动，并允许测量RL活动。注意:除非你正在使用的基材混合物，使信号持续时间延长（如双GLO的萤光素酶试剂盒购自Promega公司），使用闪光灯套件除了基板时需要快速的测量（5分钟内）。 </li></ol></li></ol>

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确定和LL作者挂起的有关复用荧光素酶技术专利。

有几个传播者基于荧光素酶报告系统（如Promega公司，瞄准系统，New England Biolabs公司，赛默飞世尔）的上线提供了有用的资源，对于那些第一次招待的高通量筛选。此外，一些优秀的评论关于优化利用高通量筛选基因的发现，以及如何基于RNAi筛查结果可以集成其他组学为基础的数据集应该是有帮助的15,16。 "点击"高通量筛选方法的选择是另一个讨论的主题是超出了本报告的范围。关于这一主题的一些评论是17,18。然而，一个重要的标准是验证转录激活的记者使用的基础上选定的从屏幕上点击的，相同的perturbagen（RNAi技术或化学）的能力验证的靶基因诱导相同的效果作为衡量采用RT-PCR，定量PCR，或微阵列分析。此外，牢记响应作为衡量记者的幅度通常高于诱导内源性记者成功合成记者的一个特点是提供了一个强有力的信号。最后，这共转染的战略优势是容易与哪一个可以迅速组装不同记者鸡尾酒，监测细胞活动所需的设定。稳定的细胞系窝藏记者使用更多的劳动力密集型的，但它可能会增加信号噪比给定的可变性反应现在主要限于单用siRNA转染效率。

监测多样化的细胞的生物过程的各种荧光素酶为基础的记者，是市售的。这些DNA构建的大部分编码荧光素酶蛋白质的诱导表达特定细胞刺激或扰动。最强大的以转录为基础的记者将表达荧光素酶的酶合成的增强子元件或建立对他们的可靠性报告生理上相关的转录事件1,2的基因的启动子区的控制之下。也有记者反荧光素酶系统，​​利用GAL4-UAS驱动荧光素酶蛋白的表达。 Gal4的是一种酵母转录激活因子，在UAS增强子的Gal4的特异性结合以激活转录的基因序列放置3向下流。这些类型的荧光素酶系统通常支持了DNA质粒 - 一个编码的GAL4融合蛋白的调控Ry的感兴趣的蛋白部分，和第二编码的控制下的一个或更多的UAS序列下的荧光素酶的蛋白质。因此，细胞的荧光素酶的信号反映感兴趣的蛋白质的活性水平。基于荧光素酶的记者的另一个常见用途是用于监测目的蛋白的融合蛋白的稳定性，即荧光素酶4。不管记者的类型，在这里指出，作为一个可以不承担任何记者的特异性已审问一个买者自负。因此，尽职调查是必需的任何研究战略纳入新记者构造。 读出的荧光素酶的选择可以是一样重要的控制其表达的DNA元件的可靠性。鉴于萤火虫荧光素酶（佛罗里达州）是最常用的酶在市售的结构，出现新的荧光素酶报告系统，不需要ATP（为的情况下佛罗里达州基反应），或者采用更稳定的酶，发出更强的信号，承诺这个研究平台，以提高效率和可靠性。荧光素酶的记者关于选择化学研究中一个重要的注意事项是佛罗里达州的易感性化学抑制，可能会引起虚假的报告。根据我们的经验，其他酶如海肾或荧光素酶Gaussia（RL和GL），分别利用腔肠基板不易抑制小分子5。 复用荧光素酶读出的最常见的格式，主要是因为可获得的易于使用的工具包，顺序地从单一样品中酶活性的测量的能力，需要使用FL和RL。在大多数试剂盒，样品第一次接触到荧光素揭示佛罗里达州的活动，然后通过淬火试剂同时沉积腔肠素产生次生次生RL信号。与另外的其他的荧光素酶可分泌，如GL或，使用另一种基片，如海萤荧光素酶（CL），更大数量的可能性，用于产生高的内容数据，使用荧光素酶已经出现。可以在这里找到6全基因组RNAi筛选使用这种技术的一个例子。这些技术进步反过来刺激特定的机制来解渴，以限制串扰7每种类型的荧光素酶的发展。在我们的手中，我们注意到一个更大的频率的"边缘效应"（莫名的趋势与边缘的高滴度文化板相关的细胞的活性），如GL或CL分泌荧光素酶。另一方面，例如分泌的荧光素酶动力学分析是有用的，因为它们使信号采样不掺细胞活力。 这里介绍我们的研究中，我们将同时监测三个癌症有关的活动细胞过程:P53，KRAS，Wnt信号转导通路。在我们的研究中成立记者pp53-TA-吕克FL记者从Clontech公司（以下简称为P53-FL记者）8，一个的记者Elk1-GAL4/UAS-CL系统（以下简称ELK-1记者安捷伦）， 8X的TCF RL记者，集成了多种合成的增强子元件Wnt信号通路的转录调控因子被称为T细胞因子（或粤港科技合作资助计划）9-11确认。

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>PathDetect Elk1 Trans-Reporting System</td><td>Agilent Technologies Inc.</td><td>219005</td><td></td></tr><tr><td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td><td>Clontech Lab Inc.</td><td>631914</td><td></td></tr><tr><td>Effectene Transfection Reagent</td><td>Qiagen Inc.</td><td>301427</td><td></td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega Corp.</td><td>E1960</td><td></td></tr><tr><td>Cypridina Luciferase Assay reagent</td><td>Targeting Systems</td><td>VLAR-1</td><td></td></tr><tr><td>HCT116 cells</td><td>ATCC</td><td>CCL-247</td><td></td></tr><tr><td>CytoTox-Fluo Cytotoxicity Assay</td><td>Promega Corp.</td><td>G9260</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>MultiFlo Microplate Dispenser</td><td>Labsystems Inc.</td><td>Model 832</td><td></td></tr><tr><td>Biomek FXP Laboratory Automation Workstation</td><td>Beckman Coulter Inc.</td><td>A31842</td><td></td></tr><tr><td>PHERAstar FS-multi-mode HTS microplate reader</td><td>BMG Labtech Inc.</td><td>0471-101A</td><td></td></tr><tr><td>Bright-Line Reichert Hemacytometers</td><td>Hausser Scientific Company</td><td>1490</td><td></td></tr></tbody></table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

使用RNA干扰（RNAi）的基因的功能基因组规模的审讯化学听话癌细胞漏洞的快速鉴定具有很大的前景。限制这一技术的潜力是无法迅速划定机理的基础表型的结果，从而告知的发展，分子靶向治疗策略。我们在这里概括的方法来解构细胞表型诱导RNAi介导的基因打靶使用多路记者关键癌症细胞相关的流程监控系统，使。高内涵筛选方法是通用的，可以很容易地适用于其他类型的大型分子库的筛选。

尽管绘制各种癌症的突变景观采用大规模的基因组测序工作12-14的进步，我们仍然不到位有一个系统的方法将这些意见纳入干预战略。结直肠癌（CRC），预测的突变，影响组件的三个细胞过程 - Wnt基因/β-catenin蛋白，P53，和KRAS信号 - 被发现在所有肿瘤的99％，这表明他们是在肠道细胞转化的关键驱动因素13。因此，癌症基因组数据集可能丰富支持共同点促癌过程的基因，并可能被利用快速识别癌细胞漏洞的。为了调查这一假说，我们设计了一套适合高通量基因功能的审讯策略，就Wnt基因/β-catenin蛋白，P53，和KRAS信号。我们首先证明特异性的每一个报告系统在单个报告以的ŗ测试（ 图1），然后提供的证据表明，这些记者可以一起使用这三个重要的细胞过程（ 图2）的基因的活性的同时测量。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50369/50369fig1highres.jpg" src="/files/ftp_upload/50369/50369fig1.jpg" /> 图1。高通量的荧光素酶为基础的检测用于监测Wnt基因/β-连环蛋白，p53和的嘉仕/ Erk通路在哺乳动物细胞中的状态。三个不同的筛选平台的鲁棒性以个别方式评估之前，复用控制siRNA靶向完善的通路的组成部分。指定的结直肠癌细胞株转8X TCF，pp53-TA吕克，或ELK-1萤火虫荧光素酶编码记者构造沿用siRNA靶向β-连环蛋白，P53，或KRAS，分别池。 Pathwa每种细胞系的y有关的基因型表示。各筛选平台的强度进行评估，这些siRNA的诱导有关的信号转导途径的影响的再现性。 β-连环蛋白的siRNA有8X TCF RKO细胞的报告基因活性没有影响，DLD-1，HCT-116细胞，分别是缺乏的APC途径抑制蛋白和β-连环蛋白的表达的活化形式。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50369/50369fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50369/50369fig2highres.jpg" src="/files/ftp_upload/50369/50369fig2.jpg" /> 图2。基于复用荧光素酶记者高含量的细胞分析基因的功能。 A.同时监测WNT /β-猫的战略示意图<html>叶宁，p53和嘉仕通路活动。佛罗里达州=萤火虫荧光素酶，RL = 海肾萤光素酶，CL = 海萤荧光素酶（分泌酶）。 FL和RL活动确定在裂解使用萤光素及腔肠基板。 CL在培养基中的活性的测量是使用光能，因而发光。其它报告系统可以被纳入这个协议来增加每个实验的内容。例如，可以利用CytoTox面粉测定用来监测细胞毒性通过采样释放到培养基中的细胞内蛋白酶的水平。除了 ​​不影响的CytoTox检测试剂活性的CL记者。B.确定特定途径的漏洞利用RNAi。使用指定的siRNA池复用荧光素酶系统中描述的"A"来量化基因对细胞功能的奉献的能力进行了测试。NK"&gt;点击这里查看大图。

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

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

实现系统级的细胞过程的理解，是现代细胞生物学的目标。我们在这里描述的各种细胞功能的复用荧光素酶记者的战略点审问基因的功能基因组规模的RNAi库。

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  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Nanyang Technological University

Research

JoVE Journal

Medicine

11.0K 次观看.  UT Southwestern Medical Center. 实现系统级的细胞过程的理解，是现代细胞生物学的目标。我们在这里描述的各种细胞功能的复用荧光素酶记者的战略点审问基因的功能基因组规模的RNAi库。

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

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

科研

癌症研究

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

用于患者来源的肿瘤类器官药物评估的自动动力学成像

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

作者聚焦:为基于类器官的药物反应分析开发多重动力学分析

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

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 ...

免疫与感染

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

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

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 ...

生物学

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

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

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此视频中的章节

基于荧光素酶的大抑癌 (拉图) 生物传感器监测河马信号通路活动

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

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

使用宽场活细胞成像进行批量和单类器官分析的多参数肿瘤类器官药物筛选

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

多重活细胞成像在患者来源的癌症类器官模型中用于药物反应

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

使用自制双辉光萤光素酶检测的高通量筛选功能

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性