笔者想感谢Karsta Luettich和Gregory Vuillaume他们的手稿的审查。

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pardaxin+an+antimicrobial+peptide,+triggers+caspase-dependent+and+ROS-mediated+apoptosis+in+HT-1080+Cells.">Pardaxin an antimicrobial peptide, triggers caspase-dependent and ROS-mediated apoptosis in HT-1080 Cells.</a> Mar. Drugs. 9 (10), 1995-2009 (2011).</li><li>Bao, S., Knoell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+modulates+cytokine-induced+lung+epithelial+cell+barrier+permeability.">Zinc modulates cytokine-induced lung epithelial cell barrier permeability.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 291 (6), L1132-L1141 (2006).</li><li>Xia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+Cytotoxicity+Profiling+Using+Quantitative+High-Throughput+Screening.">Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening.</a> Environ Health Perspect. 116 (3), 284-291 (2008).</li></ol>

所有作者都是菲莫国际的员工。菲利普莫里斯国际公司是该项目的资金和赞助商的唯一来源。

用于替代动物实验和新的高通量测试方法的需求在过去几年里得到了广泛的讨论。这已使科学家和监管当局调查标准毒性试验的替代方法，利用细胞试验紧密模仿靶组织的生理学。在这项研究中，我们已经证明结合具有高含量筛选（HCS）平台实时细胞分析仪（RTCA）评估暴露于单个CS成分对人肺上皮细胞的影响的适用性。这种设置可以类似用于评估其他各种空气污染物，空气中的颗粒，和纳米粒子诱导的细胞毒性。此外，所获得的结果可以与来自全基因组转录和计算方法基于因果生物网络相匹配。据此前报道，这种方法使我们能够证实的分子通路数据在CS曝光5与HCS终点的扰动，解决这些途径扰动也表型。 为流程图测定中，实时细胞分析提供以剂量和时间依赖性的分辨率，这允许它的剂量和暴露时间点可以是用于下游分析14有利更好的决策细胞活力相关的信息。分析仪的原理依赖于在由细胞产生的，因为它们附着和扩散上覆盖有金微电极的培养以及表面电阻抗的变化。阻抗被转换成称为小区索引一个无量纲参数，其可用于监测细胞粘附，扩散，形态和最终细胞活力。虽然该技术不提供关于细胞毒性机制的信息，其灵敏度能的，即使在非常低的剂量在所述HCS并不翔实形态学细胞变化的检测（数据未显示）。基于前雇主OU中的实验中，我们已经注意到，RTCA方法能够检测在较低剂量相比，HCS端点形态学变化。 在与实时细胞分析初步筛选中，HCS平台被用来获取关于每个HPHC引起的那种细胞毒性的更深入的信息。允许个人资料HPHCs对他们在细胞车厢潜在影响HCS检测板/细胞器以及识别那些引发遗传毒性和氧化应激。该分析揭示了不同的配置文件，从而选定HPHCs诱导NHBE细胞毒性。在一般情况下，所有的化合物中，除苯酚，发现在最高测试剂量以诱导坏死。在H2AX的癌症发展1 aminonaphtalene诱导的磷酸化的潜在作用，作为遗传毒性标志物，但是HCS面板中的线粒体毒性读出这个HPHC中还发现了活性（质量增加，细胞色素C relea一致本身）和氧化应激（GSH耗竭）。同样，如先前所描述，苯酚被确定以诱导线粒体功能障碍，并导致DNA损伤以及谷胱甘肽耗竭。铬（VI），列为I组致癌的化合物之一，和巴豆醛也都鉴定为基因毒性，特别是铬（VI）也诱导细胞凋亡（半胱天冬级联活化）和巴豆醛引起增加ROS产生。最后砷（V）中，被发现会引起cJun磷酸化这是应力激酶途径激活的标志。 在这项研究中，我们利用NHBE细胞在体外肺上皮细胞的模型。在HCS设置使用这些细胞是前所未有的，使更广泛的端点，包括遗传毒性和氧化应激标志物的调查。两个活细胞和固定细胞染色方法被我们的协议内描述的，示范的整体技术的灵活性。 f中行为，非常相同的协议可以被应用到更广泛的目标，这可以通过使用任何荧光染料或抗体的加以处理。对的活染色方案的成功执行，则尊重培养时间，因为一些染料具有有限的半衰期和荧光信号完成图像采集之前可能降低是重要的。同样重要的是要考虑到，如果使用不同的细胞类型中，所有的染色条件，应重新评估，作为最佳的染料浓度和温育时间可以是不同的。 在当前的论文中，我们描述了只有五个化合物，其中的HCS方法筛选的场景。考虑到先前描述的板布局，他们被给予超过2种不同的板集总共24板（6测定和2的时间点）板.The数量也可以增加，从而允许更多的化合物或同时筛选在不变拟多个端点的tigation。在这样做之前，但是，应该考虑到，某些端点（GSH和ROS）需要立即采集，并作为结果，板的给药应以交错的方式来执行，以允许采集先前板。另一方面，使用固定的细胞染色协议表示有利的，因为在板可堆叠，在定影后的任何步骤中断协议，用于染色过程完成在稍后阶段。这种方法，例如，将提供操作员的时间来完成所有的活细胞染色板而不损害数据质量。 通过减少板的数量进一步优化工作流程，它也将是可能的复用多个端点在一起。例如，在这种情况下DNA损伤和应激激酶可以一起研究简单地使用两个二级抗体与荧光发射的不同的Channels。所述HCS平台的不断发展，其中包括全自动细胞接种，化合物稀释，剂量和染色，以及加入的新的端点将进一步扩大HCS平台的能力，作为对上皮HPHCs和其他细胞类型的有力的分析工具。

毒理学风险评估历来依赖于使用动物模型当中，虽然在生命科学基础，也有不足之处联系，如不一致译​​人类和成本高。此外，出现了越来越多的努力，找到"3R原则"2（替代，减少和优化）的精神动物试验替代方案。这种努力已经加速，在过去的几年中，这不仅是因为最近的进展，如高通量的技术和系统生物学方法，但由于立法限制使用动物试验也，特别是在欧洲联盟。 的细胞信号途径调节到毒性损伤的响应的复杂性使得它显然使用单一毒理学终点将不足以描述的某些化合物的毒理学基础。对于这一点，数百相互作用的p的相互有助于生物网络roteins也将需要被考虑在内。研究毒物对这些网络的影响，一个系统毒理学方法与表型中，高通量筛选测定组合是有用的推断效能并同时提供对个别毒物作用机制的更多信息。 在这项研究中，我们采用HCS作为强大的筛选工具，它是由一个自动显微镜和生物软件应用的，可以采集，处理和分析来自特定的基于荧光的细胞分析而得的图像数据。这允许在小区内的视觉变化进行量化，在单细胞或亚细胞水平，并可以同时分析许多参数。3例如，DNA双链断裂是使用组蛋白H2AX的磷酸化的基于抗体的鉴定评估，并使用细胞烫发活性氧（ROS）的定量eable超敏感的染料。 因为肺上皮细胞代表针对吸入毒物，包括香烟烟雾中的第一生物屏障，我们利用主支气管上皮细胞作为体外模型来分析由美国食品和药品管理局公布HPHCs的效果。4本手稿是一个后续-最多上以前的研究5中，我们评估HPHCs的不同子集的生物学影响。 作为我们工作流以评估在体外的细胞毒性的一部分，我们最初评估选择的15 HPHC的的效力，使用基于阻抗的实时蜂窝分析使我们能够建立剂量范围，适合于随后的HCS（RTCA）系统分析（ 图1）。毒理学评估HCS然后用细胞毒性九个多参数进行端点，每次在两个时间点（4和24小时）监测。所使用的标记物指示线粒体毒性，DNA损伤，应激激酶，活性氧（ROS），谷胱甘肽（GSH）的含量，蛋白酶3 - 7活性，细胞色素C释放和细胞膜的通透性，如表1所述。 我们的方法使识别和香烟烟雾成分通过剂量和时间依赖性的采样的效果的表征。最终，这产生每个HPHC 的体外毒理学特性。多组学方法也可以被用来进一步补充HCS分析。这最后还提供了在细胞信号和/或转录水平的影响有了更深的了解。

<table>
	<tbody>
		<tr>
			<td>Cellomics ArrayScan VTI HCS Reader</td>
			<td>Thermo</td>
			<td>N01-0002B</td>
			<td></td>
		</tr>
		<tr>
			<td>xCelligence RTCA MP</td>
			<td>ACEA</td>
			<td>05331625001</td>
			<td></td>
		</tr>
		<tr>
			<td>Screener (HCS)</td>
			<td>Genedata</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY counter TTC</td>
			<td>Roche</td>
			<td>05 651 719 001</td>
			<td></td>
		</tr>
		<tr>
			<td>e-Plates VIEW 96</td>
			<td>ACEA</td>
			<td>06 472 451 001</td>
			<td></td>
		</tr>
		<tr>
			<td>RTCA Frame 96</td>
			<td>ACEA</td>
			<td>05232392001</td>
			<td></td>
		</tr>
		<tr>
			<td>RTCA Cardio Temperature Tool</td>
			<td>ACEA</td>
			<td>2801171</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealer breathseal</td>
			<td>Greiner bio-one</td>
			<td>676051</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human Bronchial Epithelial cells (NHBE)</td>
			<td>Lonza</td>
			<td>CC-2540</td>
			<td>non-smoking 60-year-old Caucasian male donor&#160;</td>
		</tr>
		<tr>
			<td>BEGM BulletKit</td>
			<td>Lonza</td>
			<td>CC-3170</td>
			<td>Warm at 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>ReagentPack Subculture Reagents kit</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td>Warm at 37 &#176;C before use</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Flask filter cap 75cm2</td>
			<td>Thermo Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well assay plate&#160; black&#160;</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Fisher Scientific</td>
			<td>PI-62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Draq5 (For Far Red Nuclear Staining)</td>
			<td>Biostatus</td>
			<td>DR50200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Dye: MitoTracker Red CMXRos</td>
			<td>Life technologies</td>
			<td>M-7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Dye: MitoTracker Red CM-H2XRos</td>
			<td>Life technologies</td>
			<td>M-7513</td>
			<td></td>
		</tr>
		<tr>
			<td>ROS Dye: Dihydroethidium</td>
			<td>Sigma</td>
			<td>D7008</td>
			<td></td>
		</tr>
		<tr>
			<td>ROS Dye: CellROX</td>
			<td>Life technologies</td>
			<td>C10422</td>
			<td></td>
		</tr>
		<tr>
			<td>ROS Dye: MitoSOX</td>
			<td>Life technologies</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH Dye: Monochlorobimane</td>
			<td>Sigma</td>
			<td>69899</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>GSH Dye: Monobromobimane</td>
			<td>Life technologies</td>
			<td>M-1378</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Membrane permeability Dye: YO-PRO-1&#160;</td>
			<td>Life technologies</td>
			<td>Y3603</td>
			<td>Irritating&#160;</td>
		</tr>
		<tr>
			<td>Membrane permeability Dye: TO-PRO-1&#160;</td>
			<td>Life technologies</td>
			<td>T3602</td>
			<td>Irritating&#160;</td>
		</tr>
		<tr>
			<td>Membrane permeability Dye: TOTO-1&#160;</td>
			<td>Life technologies</td>
			<td>T3600</td>
			<td>Irritating&#160;</td>
		</tr>
		<tr>
			<td>Caspase Dye: Cellevent Caspase 3/7 green</td>
			<td>Life technologies</td>
			<td>C10423</td>
			<td>Irritating&#160;</td>
		</tr>
		<tr>
			<td>Anti-Cytochrome C antibody (Mouse)</td>
			<td>Thermo</td>
			<td>MA5-11823</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-c-Jun antibody (Mouse)</td>
			<td>Thermo</td>
			<td>MA5-15889</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-phospho-H2AX antibody (Mouse)</td>
			<td>Thermo</td>
			<td>MA1-2022</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG DyLight 650&#160;</td>
			<td>Abcam</td>
			<td>ab96878</td>
			<td></td>
		</tr>
		<tr>
			<td>10X permeabilization buffer</td>
			<td>Fisher</td>
			<td>8408400</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Formaldehyde solution</td>
			<td>Sigma</td>
			<td>F1635</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>10X blocking buffer</td>
			<td>Fisher</td>
			<td>8408500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline&#160;</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>Sigma</td>
			<td>S4400</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Valinomycin</td>
			<td>Sigma</td>
			<td>V0627</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td>Sigma</td>
			<td>36541</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Anisomycin</td>
			<td>Sigma</td>
			<td>A9789</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Ethacrynic acid</td>
			<td>Sigma</td>
			<td>E4754</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>1-Aminonaphthalene</td>
			<td>Sigma</td>
			<td>34390</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>2-Nitropropane</td>
			<td>Sigma</td>
			<td>130265</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Acetamide</td>
			<td>Sigma</td>
			<td>695122</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>650501</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma</td>
			<td>A9099</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Arsenic (V)</td>
			<td>Sigma</td>
			<td>A6756</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Benzene</td>
			<td>Sigma</td>
			<td>12540</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Chromium (VI)</td>
			<td>Sigma</td>
			<td>216623</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Crotonaldehyde</td>
			<td>Sigma</td>
			<td>262668</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Methyl ethyl ketone</td>
			<td>Sigma</td>
			<td>34861</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Nickel&#160;</td>
			<td>Sigma</td>
			<td>203866</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Nitrobenzene</td>
			<td>Sigma</td>
			<td>48547</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma</td>
			<td>P5566</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Quinoline</td>
			<td>Sigma</td>
			<td>241571</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma</td>
			<td>34866</td>
			<td>Toxic</td>
		</tr>
	</tbody>
</table>

high content screening analysis to evaluate the toxicological effects of harmful and potentially harmful constituents (hphc)

香烟烟雾（CS）是心血管和肺疾病的主要危险因素。因为CS是一个包含7000多化学品1这是具有挑战性的评估个别成分其总体毒性的贡献复杂的气溶胶。个别成分以及混合物的毒理学配置文件可以然而，建立了体外 ，由通量筛选工具，这使烟草烟雾的危害和潜在的有害成分（HPHCs）的分析施加高，受美国食品和药品的定义管理局（FDA）。2 对于初步评估，使用该化合物的毒性的实时，无标记评估的基于阻抗的工具。该仪器读数依赖于细胞黏附，活力和形态，所有共同提供的电池状态的概述。无量纲参数，命名的单元格指数，用来量化。一组的DIF同的染色方法是为基于荧光成像开发的调查和HCS平台被用来获取对每个HPHC引起的一种细胞毒性更深入的信息。 测试的15个成分中，只有五人入选基于HCS-分析，他们注册了一个可计算的LD 50（&lt;20毫米）。这些包括1- aminonaphtalene，砷（V），铬（VI），巴豆醛和苯酚。基于它们在HCS效果，1- aminonaphtalene和酚可确定以诱导线粒体功能障碍，并连同铬（VI），为基于增加组蛋白H2AX的磷酸化基因毒性。巴豆醛被确定为氧化应激诱导剂和砷作为应力激酶途径的活化剂。 这项研究表明，阻抗和基于HCS技术的组合提供了用于在体外的CS成分的评估一个强大的工具。

RTCA 因为没有检测到毒性作用，当HCS端点不会信息，没有显示的那些化合物细胞活力下降到在RCTA不是由HCS（ 图3b，C，D，G，K，L，M所测试的最高浓度页 ）。化合物显示仅在最高浓度细胞活力下降（ 图3e，O）也被取消了HCS。最后，只用一个可计算的LD 50（&lt;20毫摩尔）的成分被选择用于进一步的HCS分析（ 图 3a，F，H，J，N）。符合上述标准HPHCs是:1-aminonaphtalene，砷（V），铬（VI），巴豆醛和苯酚。 HCS 作为质量检查（QC），阳性对照是科幻首先进行分析，以保证正确地进行该染色步骤。阳性对照处理的细胞的代表性图片示于图6中 ，数据值是如前所述标准化为车辆。没有剂量反应曲线被绘制为只有三个剂量测试，不是所有的三个剂量都在每一个时间点考虑。阳性对照的剂量被选择（基于先前的实验中，未示出的数据），使适当的反应是为在两个4小时和24小时的每个端点观察。在特定的剂量1和2中使用而剂量2和3用于评估在24小时的效果，在4小时来评估的效果。如果为阳性对照的剂量没有观察到反应板被丢弃。注意，对于所有的端点，除非线粒体膜电位和GSH含量时，信号强度的增加，预计。 所有的化合物，除苯酚，引起坏死性苯氧<html>型的基础上，增加细胞膜的通透性（ 图7a，F，H，1）。 1- aminonaphtalene，铬（VI），巴豆醛和苯酚被确定为基础上，增加组蛋白H2AX的磷酸化（ 图7E，J，N，P）为基因毒性。苯酚和1- aminonaphtalene被发现诱发严重线粒体功能障碍（ 图7b，邻 ），其与1-aminonaphtalene，导致了增加的细胞色素C的释放（ 图7c）。增加的胱天蛋白酶3/7活性的检测在铬曝光提供凋亡事件的证据。氧化应激诱导（ROS或GSH）也处理后与1- aminonaphtalene，巴豆醛和苯酚（ 图7d，M，Q）进行检测。最后，砷诱导细胞应激的转录因子cJun（ 图7克 ）增加的磷酸化证明。 负载/ 53987 / 53987fig1.jpg"/&gt; 图1.化合物TOX-探查工作流。 一个）的工作流程的示意图，随后在这项研究。首先，使用RTCA平台来选择适当的剂量为后续HCS来表征特定化合物的毒性特征。b）该研究的实验设计进行剂量范围发现。播种后24小时，细胞剂量和阻抗值在接下来的24小时连续监控，而HCS终点进行了调查4和给药后24小时。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53987/53987fig2.jpg" /> 图2. RTCA曝光板。首先被执行五步1点10连续稀释生成的复合主盘。每种化合物，包括车辆控制（剂量0），然后在一式三份与介质和星形孢菌素作为对照加入到曝光板在一起。需要注意的是序列传输时保持剂量，最高剂量是行号1，而车辆控制是行数7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53987/53987fig3.jpg" /> 图3代表RTCA细胞活性的结果。a） 第 1-aminonaphtalene， 二）2-硝基丙烷， 三）乙酰胺， 四）丙酮， 电子）丙烯酰胺，f）的砷（V）中，G）苯，h）的铬（VI） ，j）的巴豆醛，k）的甲基乙基酮， 升）NickeL（II）中，m）硝基苯，n）的苯酚， 邻）喹啉， 对）甲苯。在24小时的血样，曲线（AUC）下面积计算各剂量（包括阳性对照和载体）从0到100％的活性（y轴），其中0反映的活性和标准化的范围内车辆与阳性对照-100。然后值描绘，并用四参数Hill方程，并在可能，LD 50计算拟合。浓度对数标度（X轴）表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53987/53987fig4.jpg" /> 图4.稀释方案为HCS试验得到阳性对照化合物A）阳性对照的加入和已经hicle到串行稀释板。b）该阳性对照的系列稀释。 三）200X阳性对照的剂量。 德）的200倍阳性对照的剂量在介质（1:40稀释），产生含有5倍剂量阳性对照板。请注意，每个剂量是一式三份稀释，以反映在曝光板的最后布局）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53987/53987fig5.jpg" /> 图5. HCS曝光板。首先被执行五步稀释生成复合主盘。每个化合物，包括车辆控制（剂量0），然后在一式三份与阳性对照加入到曝光板在一起。请注意剂量订单转移后保持，最高剂量是行号1，而车辆控制是行数7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53987/53987fig6.jpg" /> 图6.代表性的荧光抗体照片或染料染色的细胞的 ） 核参数 - 核染料 :它与DNA结合的活的或固定细胞中的渗透染料。此染色剂用于识别各个细胞标记的核区域b） 坏死 - 细胞膜渗透性染料 :的细胞膜完整性的基于染料的检测。试剂在本质上是不可渗透的细胞膜。期间坏死，膜变得可渗透和染料进入细胞并与DNA结合产生强光S。ignal 三） 凋亡 - 细胞色素C:细胞色素C释放，早期凋亡的一个著名标志性的基于抗体的检测。在细胞凋亡的诱导，细胞色素C从线粒体释放并扩散到细胞核四）DNA 损伤 - pH2AX:组蛋白H2AX的磷酸化的基于抗体的检测，双链DNA断裂的一个公知的标志E） 胁迫激酶 - cJun:在丝氨酸73 cJun的，细胞应激的一个公知的标志的磷酸化的基于抗体的检测六） 氧化应激 - DHE:超氧自由基的基于染料的检测。本身二氢乙荧光在细胞质蓝色，而氧化型荧光乙锭在红DNA嵌入G）GSH - MBCL:免费GSH分子染料检测。 MBCL与谷胱甘肽反应来产生高荧光产品高） 细胞凋亡 - 胱天蛋白酶激活3/7:3/7蛋白酶的活性基染料检测。试剂是非荧光与抑制DNA结合一个4个氨基酸的肽。在胱天蛋白酶3/7的活化，该肽被裂解使染料与DNA结合，并产生一个明亮，荧光响应。面板BH显示阳性对照处理的细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53987/53987fig7.jpg" /> 图7.代表性的HCS结果:1 aminonaphtalene（AE），砷（V）（f和g），铬（VI）（ 香港 ），巴豆醛（LN） ​​和苯酚（OQ）。 4小时（蓝线）和24小时（橙色线）信号计算各剂量和归一化到车辆活性（0％）。这不包括在曲线拟合计算值显示为灰色。浓度对数标度（X轴）表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53987/53987fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>化验</td><td>端点＃ </td><td>生物终点</td><td>细胞室</td><td>输出功能</td></tr><tr><td rowspan="4">细胞毒性</td><td> 1 </td><td>线粒体质量6 </td><td>细胞质</td><td>现货平均面积</td></tr><tr><td> 2 </td><td>线粒体膜电位6 </td><td>细胞质</td><td>现货平均强度</td></tr><tr><td> 3 </td><td>细胞色素c释放<sup&gt; 7 </td><td>核</td><td>平均强度</td></tr><tr><td> 4 </td><td>细胞膜的通透性8 </td><td>核</td><td>平均强度</td></tr><tr><td> DNA损伤</td><td>五</td><td>磷酸H2AX 9 </td><td>核</td><td>平均强度</td></tr><tr><td>应激激酶</td><td> 6 </td><td>磷酸cJun 10 </td><td>核</td><td>平均强度</td></tr><tr><td> ROS </td><td> 7 </td><td> ROS 11 </td><td>核</td><td>平均强度</td></tr><tr><td> GSH含量</td><td> 8 </td><td> GSH 12 </td><td>细胞质</td><td>现货平均强度</td></tr><tr><td>细胞凋亡</td><td> 9 </td><td>胱天蛋白酶3 13 </td><td>细胞质</td><td>现货平均强度</td></tr></tbody></table> 表1. HCS一个列表ssays和端点。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td><td rowspan="2">车辆</td><td colspan="6" rowspan="2"> RTCA剂量（μM） </td><td rowspan="2"> LD 50 </td><td colspan="5"> HCS剂量</td></tr><tr><td colspan="2"></td><td colspan="4">细胞活力选定（​​μM） </td><td> 3R4F（NM） </td></tr><tr><td colspan="2"> 1 Aminonaphtalene </td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 280μM </td><td> 2000 </td><td> 500 </td><td> 200 </td><td> 150 </td><td> 0.27 </td></tr><tr><td colspan="2"> 2-硝基丙烷</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">乙酰胺</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">丙酮</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">丙烯酰胺</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">砷（V） </td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 160μM </td><td> 200 </td><td> 100 </td><td> 50 </td><td> 25 </td> &lt;TD&gt; 0.17 </td></tr><tr><td colspan="2">苯</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">铬（VI） </td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 20μM </td><td> 100 </td><td> 50 </td><td> 20 </td><td> 10 </td><td> 0.06 </td></tr><tr><td colspan="2">巴豆醛</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 200μM </td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2000 </td></tr><tr><td colspan="2">甲乙酮</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt;20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">镍</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">硝基苯</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">苯酚</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> 2300μM </td><td> 5000 </td><td> 2000 </td><td> 1000 </td><td> 500 </td><td> 240 </td></tr><tr><td colspan="2">喹啉</td><td>乙醇</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td&gt; <td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="2">甲苯</td><td>水</td><td> 20000 </td><td> 2000 </td><td> 200 </td><td> 20 </td><td> 2 </td><td> 0.2 </td><td> &gt; 20毫米</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表2. 在治疗24小时 相对LD 50 测试HPHC化合物列表 。选择HCS分析化合物是橙色高亮显示和测试的剂量也给予。该3R4F剂量相当于构成本，在一个棒的从基准香烟3R4F烟雾的量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>化验</td><td>复合</td><td>原液</td><td>溶剂</td&gt; <td colspan="3">剂量（多个）（μM） </td></tr><tr><td>细胞活力</td><td>星形孢菌素</td><td> 10毫</td><td> DMSO </td><td colspan="3"> 50 </td></tr><tr><td>细胞毒性</td><td>霉素</td><td> 10毫</td><td> DMSO </td><td> 50 </td><td> 20 </td><td>五</td></tr><tr><td> DNA损伤</td><td>百草枯</td><td> 100毫米</td><td> DMSO </td><td> 500 </td><td> 200 </td><td> 50 </td></tr><tr><td>应激激酶</td><td>茴香</td><td> 2毫米</td><td> DMSO </td><td> 10 </td><td> 4 </td><td> 1 </td></tr><tr><td> ROS </td><td>鱼藤酮</td><td> 200毫米</td><td> DMSO </td><td> 1000 </td><td> 400 </td><td> 100 </td></tr><tr><td> GSH含量</td><td>利尿酸</td><td> 200毫米</td><td> DMSO </td><td> 1000 </td><td> 400 </td><td> 100 </td></tr><tr><td>细胞凋亡</td><td>星形孢菌素</td><td> 40毫米</td><td> DMSO </td><td> 200 &lt;/ TD&gt; <td> 50 </td><td> 20 </td></tr></tbody></table> 表3列出用于每个检测阳性对照和浓度。

Watch this Scientific Journal Video about High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC at JoVE.com

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

high-content-screening-analysis-to-evaluate-toxicological-effects

Nanyang Technological University

Research

JoVE Journal

Biology

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

12.1K 次观看.  Philip Morris International R&D. The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds. 

视频: High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

毒理学化验一个后生药物对疟蚊的发育，繁殖和存活率测试效果

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

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