这项工作是由蒙特利尔大学提供的默克Frosst启动资金支持。我们愿在免疫与癌症研究所研究从高通量平台，感谢博士让Duchaine和Dominic Salois他们的讨论，发表意见和反馈。 Moutih Rafei持有全宗德拉RECHERCHE EN桑特魁北克少年1奖。

所有动物方案都被蒙特利尔大学的动物管理委员会批准。小鼠通过CO 2的渐进吸入安乐死，直到没有生命体征观察颈椎脱位。该程序是由经过认证的人员进行，以确保动物以人道的方式实施安乐死，并根据加拿大议会关于动物保护的建议。 1.脾细胞培养基和流式细胞仪缓冲液的制备<ol><li>执行70％的乙醇，生物清洗引擎盖下的所有步骤。 </li><li>取出70毫升预热罗斯威尔公园纪念研究所（RPMI）1640 1X培养基（市售，并在过滤500毫升体积的无菌瓶提供），置于无菌管。除去的介质将在后面的协议被使用。 </li><li>补充剩余的430毫升的RPMI 1640 1X用50ml灭活的小牛血清（FBS），5毫升青霉素/链霉素，5毫升HEPES，将5ml非必需氨基酸，5毫升丙酮酸钠，和0.05毫升的过滤（1M）2-巯基乙醇。 注:使用水浴在37ºC设置，以避免热量惊爆细胞预温脾网上平台。这是很重要的，以避免细胞凋亡的诱导。 </li><li>为了制备流式细胞缓冲液，加入2毫升胎牛血清的98毫升磷酸盐缓冲盐水（PBS），并保持冷藏直到使用（三级优选在天）。 </li></ol> 2.从一代GFP Nur77鼠标脾脾脏细胞悬浮<ol><li>一个septically隔离从6-8周大的雌性Nur77 GFP小鼠脾脏。 <ol><li>为了实现这一目标，在无菌罩工作。浸泡在70％乙醇的牺牲鼠标的皮毛，剪刀和镊子。 </li><li>莱在其右侧的鼠标，切左上腹部象限皮肤和肌肉。检查切口面积明显定位脾然后切出来。保留脾脏在冰上直至准备脾介质以进行下一步骤。 </li></ol></li><li>放置脾（多个）在含有5ml预热脾细胞培养基的10cm 2的细胞培养物的培养皿。 </li><li>醪使用无菌注射器柱塞直到溶液是浑浊的脾脏。脾胶原基质应保持该过程的结束。 </li><li>以下在脾细胞中的广泛糖化，收集细胞悬浮液，并使之通过放置在一个50毫升管过滤出任何碎屑或血块70微米的细胞滤网。 </li><li>离心在500×g离心5分钟。丢弃上清液后，再悬浮在2-3毫升细胞沉淀在内部产生的或商业的红细胞溶解缓冲液中。继移液（2-3倍），让悬挂休息20-30秒。 </li><li>加入5毫升的PBS或选择合适的缓冲液，然后离心细胞悬浮液在500×5分钟g。 </li><li>除去上清液（应该是红色，因为它包含裂解红Blood细胞）并重新悬浮在2毫升脾细胞中的细胞沉淀。 </li><li>使用血细胞计数器，计数用锥虫蓝染色的细胞的数目存活和死亡（坏死）细胞之间进行区分。 </li></ol> 3.从脾细胞悬浮细胞的T细胞分离<ol><li>离心细胞悬浮液5分钟，在500×g下。重新悬浮于无血清的RPMI培养基中的细胞（来自步骤1.3 50ml）中，得到10×10 6个细胞/ mL的细胞浓度。 </li><li>转移细胞以5毫升聚苯乙烯管中并搁置脾细胞的纯度评估/比较的100微升等分试样在纯化步骤结束。 </li><li>正常大鼠血清添加至50μL/ mL的细胞悬浮液，接着为T细胞的分离抗体混合物（50μL/ mL）中。 </li><li>混合细胞悬液，让它休息10分钟。这个步骤可以使抗体的鸡尾酒来约束所有不需要的细胞。 </li><li>添加链霉快速球（磁铁集成电路珠）为2.5分钟，然后用无血清培养基使体积至2.5毫升。 </li><li>放置管在3分钟的细胞隔离磁铁。 </li><li>握住磁铁和一个移动浇注出来的溶液转移T细胞悬液注入了新的聚苯乙烯管。 注:将分离的悬浮液含有纯化的T细胞。所有不想要的细胞被保持在结合到磁性链霉珠的管的一侧。 </li><li> 使用计数台盼蓝和血球分离的T细胞。 注:在这一步骤中分离的T细胞的纯度可通过前，后通过流式细胞仪的T细胞分离分析的CD3 +事件的百分比进行验证（可选）。 13 <ol><li>简要地说，离心在500xg1毫升脾细胞悬浮液。弃上清，悬浮于流式细胞仪缓冲液将细胞以1×10 6个细胞/ mL的浓度。 </li><li>添加荧光标记的抗谅解备忘录100:以1:1的浓度ËCD3抗体。孵育在4℃下30分钟。洗一次离心机在通过流式细胞分析流式细胞缓冲（400μL）重新被挂起。 </li></ol></li></ol> 4.从脾细胞悬浮细胞B细胞分离<ol><li> 遵循相同的协议，作为T细胞（步骤3.1-3.8），但使用的是B细胞分离试剂盒说明。对于纯度评价，使用CD19抗体，而不是CD3抗体。 13 <ol><li>对于纯度评估（选配），使用CD19抗体，而不是CD3抗体。离心机1毫升，在500×g下脾细胞悬浮液。弃上清，悬浮于流式细胞仪缓冲液将细胞以1×10 6个细胞/ ml的浓度。 </li><li>添加荧光标记的抗小鼠CD19抗体以1:1的浓度:100，并在4℃下孵育30分钟。洗一次，离心并在流式细胞仪缓冲液（400微升）FO重新悬浮通过流式细胞仪分析ř。 </li></ol></li></ol> 5. T细胞活化和GFP表达的诱导<ol><li>种子的T细胞，在悬浮液中，成圆底96孔板以2.5×10 5个细胞的浓度/孔。 </li><li>添加重组白细胞介素（IL）-7在2毫微克/毫升和CD3 / CD28磁珠（25微升/ 10 6个细胞）。保持T细胞未用珠，作为购买的量度代表非激活的T细胞的阴性对照的一部分。 </li><li> 12小时后，通过在每个轻轻吹打细胞悬液以及上下打破任何珠 - 细胞复合/聚集体收获从96孔板的T细胞。收集从所有孔中成5毫升聚苯乙烯管中的悬浮液中。 </li><li>放置装有用于T细胞或B细胞的纯化先前使用的相同的细胞分离磁铁内的悬浮液的管子，并允许它休息5分钟。 </li><li>转移T细胞悬液INT握住磁铁和一招涌出来解决OA新的聚苯乙烯管。 </li><li>离心细胞，在500×g离心5分钟。重悬在新鲜脾细胞培养基中的细胞以获得2×10 6个细胞/ mL的浓度。 </li><li>评估与非活化的T细胞相比通过在12到24小时后刺激流式细胞仪（可选为质量控制）GFP表达强度。 12 注:绿色荧光是内在的Nur77 GFP的T细胞，并于TCR的成功激活就体现出来了。 12 </li><li>用于通过显微镜活力和活化（可选）的评估，染色活化的细胞用Hoechst 30分钟分析前以0.2微克/ ml的浓度。添加细胞悬浮液的适当体积的盖滑动或一个平底黑色双面384孔板的孔中，并在荧光显微镜下检查，以评估活细胞。死皮细胞不会保留核武器染色。 14 </li></ol> 6. B细胞活化和GFP表达的诱导<ol><li>使用T-25培养烧瓶中，将重悬的分离的B细胞以1×10 6个细胞/ mL。 </li><li>添加10微升/毫升的抗小鼠IgG / IgM抗体（H + L）和重组CD40L在200纳克/毫升的浓度。保持B细胞未处理，作为以后测量的阴性对照的一部分（代表非活化的B细胞）。 </li><li>评估与非活化的B细胞相比在12或24小时后的刺激通过流式细胞术GFP表达强度。 注:绿色荧光是内在的Nur77 GFP B细胞和经BCR的成功激活就体现出来了。 12 </li><li>用于通过显微镜活力和活化（可选）的评估，染色活化的细胞用Hoechst 30分钟分析前以0.2微克/毫升的浓度。添加足够量的细胞悬浮液以盖滑动或一个平底黑色双面384孔板的孔中，并检查在荧光显微镜下以评估活细胞。死皮细胞不会保留核染色。 14 </li></ol>小分子7.高通量筛选<ol><li>以2×10 6个细胞/ mL的激活的T细胞或B细胞的（磁珠或抗体/ CD40L）或非活化基制备细胞悬浮液。 </li><li>板75000细胞/孔在384孔板（40微升的体积）。使用平底黑色双面384孔板或用于通过使用赫斯特染色荧光显微镜（HTS之前可选质量控制步骤）活性和激活的评估在显微镜盖玻片（参照步骤5.8和6.4的详细信息）。 14 </li><li>手动或使用自动化系统，加选择（溶于0.5％DMSO）中的药物到每个孔中。 </li><li>的载体（DMSO）添加到正（激活）和负（非ACTI氧基团）对照孔。调整DMSO浓度为最多0.5％。 </li><li>孵育所述板在37ºC24小时（或培养选择的时间）和5％ 的 CO 2。 </li><li>对筛选的当天，淡化的Hoechst 33342染色溶液（1:3333;对于如在384孔板中添加10μL的向细胞以产生总体积为50μl）。通过加入Hoechst的解决方案，以实现0.2微克/ mL的浓度染色的GFP分析前30分钟。 </li><li>轻轻吸取细胞上下以获得每一个均匀分布良好。 注意:此步骤是在使用自动系统作为细胞倾向于在井一侧积累，相反的流动方向分配所述药物（步骤7.3）的情况下，非常重要的。 </li><li>旋板在45 XG在室温下3分钟。 </li><li>离开板，以其余为在室温下15分钟。 </li><li>执行板（S）读数，使用自动化共焦含量高SCRE效果图创作（HCS）系统。 15 加载板到机器。在40X或更高的放大倍率设定的目标。使用相机＃4赫斯特（紫外线灯）和GFP相机＃1（激光488）。设置机器读取每孔6-10场。调整机器在488纳米（阅读GFP）和紫外线（读赫斯特）做每场两个连续的读数。在40X或更高的放大倍率设定的目标。 注:该系统是不需要任何调整计算机化的显微镜。焦距，入射光的强度和暴露时间是由机器自动所有的配置。 </li></ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+chemosensitivity+assays:+an+overview.">Cellular chemosensitivity assays: an overview.</a> Methods Mol Biol. 731, 219-236 (2011).</li><li>Huh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-based+system+for+screening+hair+growth-promoting+agents.">A cell-based system for screening hair growth-promoting agents.</a> Arch Dermatol Res. 301 (5), 381-385 (2009).</li><li>Weiss, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+signal+transduction:+opening+the+black+box.">TCR signal transduction: opening the black box.</a> J Immunol. 183 (8), 4821-4827 (2009).</li><li>Noel, P. J., Boise, L. H., Green, J. M., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+costimulation+prevents+cell+death+during+primary+T+cell+activation.">CD28 costimulation prevents cell death during primary T cell activation.</a> J Immunol. 157 (2), 636-642 (1996).</li><li>Michels, A. W., 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=Structure-based+selection+of+small+molecules+to+alter+allele-specific+MHC+class+II+antigen+presentation.">Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation.</a> J Immunol. 187 (11), 5921-5930 (2011).</li></ol>

作者宣称，他们没有竞争的经济利益。

数读出的方法已被开发用于灵敏和可靠的HTS测定法的发展。这些措施包括色度，发光或荧光方法。虽然比色法是简单的设置，它们需要的化学品的多次加入，其可干扰或中断所测试的细胞。 23此外，它们不作为药理学作用是在特定端点评估允许生物应答的动态评估。此外，如果使用自动的HTS此方法可能需要昂贵的机器人手臂。这些因素使色度读奏与HTS的目标相容性较差，由于其?...

高通量筛选（HTS）是一个久经考验的战略新的治疗分子的鉴定或为新的医学指征FDA批准的药物重新定位广泛采用。 1到目前为止，取得HTS成功可以通过的先前发现的药物过多测定。例如，用于乳腺癌，西他列汀的治疗酪氨酸激酶抑制剂拉帕替尼;一个二肽基肽-4（DPP-4）抑制剂用作抗高血糖药物，以及用于治疗慢性髓细胞性白血病的治疗口服的Bcr-Abl的酪氨酸激酶抑制剂达沙替代表批准的药物最初由发现的一长列的几个例子HTS。 2虽然制药业的生产力近来从缺乏的新化学实体的发现遭受的，成功的药物发现的可能性可以通过增加在临床前念珠菌的数量提高TES显示调节生物学/生物化学特性。因此，适于表型筛选新HTS测定法的发展可以提供为新药命中的发现提供了重要的药理学工具的潜力。 3，4，5，6此外，高温超导现在可以以更快的速度，由于近年来显著技术改造包括定制设计的灵活的机器人装置，新颖读出的技术和大量的小型化进行。 2,7在有助于在使用表型筛选（又名向前药理学）的日益增长的兴趣的因素是人们认为着眼于功能效应，而不是关于分子靶（基于目标的筛选/生化反应）过于简化的还原假设是更有可能以翔<html>瓦特临床疗效。因此，表型筛选持有的承诺，发现新的潜在的治疗化合物和目前无法治疗的疾病的分子途径。 2 适当地识别一个给定的分子靶标或细胞功能抑制剂或活化剂，高度敏感的，可靠的检测是必需的，以便真正命中和误报之间进行区分。那么，是什么使一个良好的试验？一个给定的测定法的质量必须由信噪比（通过为Z因子反射的）首先判断。 8其次，有针对性的效果或屏幕的目标应明确规定。例如，功能性的基于细胞的方法可以用于受体的筛选提供显著优点，而不是专门设计来评估配体 - 受体结合的测定法。这样做的原因是，在后一种方法不能激动剂和拮抗剂配体之间区分。SS ="外部参照"&gt; 9相反，基于细胞的方法很可能是作为受体功能可以在一个生物的表型被直接评估更为有效（增殖，细胞周期停滞，细胞凋亡，和/或分化）。然而，必须指出的是，生物化学测定可在表型测定中提供显著优点，因为它们违反在一个特定的细胞内靶标。一个精心优化的生化检测通常会有比表型筛选数据散射少，同时简化后的相关行动的药物分子机制的研究。然而，目标为基础的或生物化学分析的主要缺点是扩增在生物系统（最初在生物化学测定法研究了特异性的损失）测试时，可能会影响非特定目标的假阳性命中率的机会。 10虽然阴性和阳性之间命中一个完善的分界点可以最大限度地减少误报的数量我n中的初步筛选，使用了生理学相关的系统模拟天然细胞环境如完整细胞，完整的组织或整个动物仍然测定设计钟摆的核心。因此，表型筛选使铅发现与对于没有标识的药物靶标疾病期望生物/表型效应，而无需化合物的活性或作用模式的先验知识。 11 在本文中研究涉及基于两个重要部件的优化和可再现的表型筛选的开发和测试:市售小鼠模型和化学化合物的聚集亚家族。对于动物模型，测定法依赖于在其中绿色荧光蛋白 （GFP）的表达是由第驱动用来自小鼠品系（Nur77 GFP）带有含一个盒的细菌人工染色体衍生的淋巴细胞ËNur77子。 12这种刺激的标志是基于这样的事实，Nur77是立即早期基因上调下列T细胞受体（TCR）或B细胞受体（BCR）刺激。 12对于筛选方法本身，使用的方法，以帮助避免琐碎类似物的筛选，同时最小化，以评估一个大化学文库（&gt; 10 5的化合物）所需的时间。这样做，通过使用虚拟筛选工具药物化学家选择的化学化合物的数据库利用来识别使用已知活性种子的结构作为参考拓扑类似的化合物。这个方法允许我们筛选较136000化学实体的整体库4398的化合物。

<table><tbody><tr><td>Nur77&lt;sup&gt;GFP&lt;/sup&gt; mice</td><td>The Jackson Laboratory</td><td>Mouse strain No. 016617</td><td>An in house colony was established at our animal facility&amp;nbsp;</td></tr><tr><td>96 wells-U culture plates, sterile</td><td>VWR International</td><td>10062-902</td><td>T-cell activation using the magnetic beads</td></tr><tr><td>70 &amp;micro;m cell strainer, sterile</td><td>Corning Inc.</td><td>352350</td><td>Generation of splenocytes cell suspension</td></tr><tr><td>5 ml polystyrene round bottom tubes, sterile</td><td>Corning Inc.</td><td>352058</td><td>Generation of splenocytes cell suspension</td></tr><tr><td>50 ml polypropylene conical bottom tubes, sterile</td><td>VWR International</td><td>89039-656&amp;nbsp;</td><td>Generation of splenocytes cell suspension</td></tr><tr><td>10 ml syringe without needle, sterile</td><td>Becton, Dickinson and Company</td><td>305482</td><td>To mash the spleen</td></tr><tr><td>T-25 culture flask</td><td>Greiner Bio-One</td><td>690 175</td><td>To incudabte B cells during activation</td></tr><tr><td>5 ml cell culture dish, sterile</td><td>Greiner Bio-One</td><td>627 160</td><td>To mash the spleen</td></tr><tr><td>Penicillin- Streptomycin (10,000 U/mL)</td><td>WISENT Inc.</td><td>450-200-EL&amp;nbsp;&amp;nbsp;</td><td>Component of the splenocyte media</td></tr><tr><td>RPMI 1600 with sodium bicarbonate and L- glutamine</td><td>WISENT Inc.</td><td>350-002-CL&amp;nbsp;</td><td>Component of the splenocyte media</td></tr><tr><td>MEM non-essential amino acids</td><td>WISENT Inc.</td><td>321-010-EL&amp;nbsp;</td><td>Component of the splenocyte media</td></tr><tr><td>HEPES free acid 1 M</td><td>WISENT Inc.</td><td>330-050-EL&amp;nbsp;</td><td>Component of the splenocyte media</td></tr><tr><td>Sodium pyruvate solution (100mM)</td><td>WISENT Inc.</td><td>600-110-EL&amp;nbsp;</td><td>Component of the splenocyte media</td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>WISENT Inc.</td><td>080-910&amp;nbsp;</td><td>Component of the splenocyte media and flow-cytometry buffer</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>WISENT Inc.</td><td>311-010-CL</td><td>Component of flow-cytometry buffer</td></tr><tr><td>2-Mercaptoethanol (55 mM)</td><td>Thermo Fisher Scientific</td><td>21985-023</td><td>Component of the splenocyte media</td></tr><tr><td>T-Cells isolation kit</td><td>Stemcell Technologies</td><td>19851</td><td>To isolate T cells</td></tr><tr><td>B-Cells isolation kit</td><td>Stemcell Technologies</td><td>19854</td><td>To isolate B cells</td></tr><tr><td>Mouse T-Activator CD3/CD28 superparamagnetic beads</td><td>Thermo Fisher Scientific</td><td>11452D</td><td>To activate T cells&amp;nbsp;</td></tr><tr><td>Cell isolation magnet</td><td>Stemcell Technologies</td><td>18000</td><td>To isolate T cells and remove the magnetic beads&amp;nbsp;</td></tr><tr><td>AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Goat Anti-Mouse IgG + IgM (H+L)</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>115-006-068</td><td>To stimulate B cells</td></tr><tr><td>Recombinant Murine IL-7</td><td>Peprotech</td><td>217-17&amp;nbsp;</td><td>To support T-cell survival during activation</td></tr><tr><td>Recombinant CD40L</td><td>R&amp;amp;D Systems</td><td>8230-CL/CF</td><td>To stimulate B cells</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>BD Pharmingen</td><td>561799</td><td>To stain T cells for flow-cytometry</td></tr><tr><td>Anti-mouse CD19 antibody</td><td>BD Pharmingen</td><td>553786</td><td>To stain B cells for flow-cytometry</td></tr><tr><td>Biomek FX&lt;sup&gt;p&lt;/sup&gt;</td><td>PerkinElmer Inc.</td><td>A31842</td><td>To re-suspend cells after 24 h incubation</td></tr><tr><td>Opera Phenix High Content Screening System</td><td>PerkinElmer Inc.</td><td>HH14000000</td><td>To analyze GFP/Hoechst signal</td></tr></tbody></table>

a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules

高通量筛选（HTS）是目前能够调节的生化反应，或细胞过程的化学实体的标识支柱。随着生物技术的进步和小分子的高潜力转化，一批新药研发创新方法的演变，这也解释了在使用HTS的复苏兴趣。肿瘤学领域是目前药物筛选中最活跃的研究领域，与靶向移植相关并发症或自身免疫性疾病的新免疫调节化合物的鉴定没有取得重大突破。在这里，我们提出体外鼠基于荧光检测淋巴细胞一种新型容易地适应新的免疫调节化合物的鉴定。这种测定法使用从转基因小鼠，其中该Nur77启动子后的T细胞或B细胞受体刺激驱动GFP的表达衍生的T或B细胞。由于GFP强度反映了靶细胞的活化/转录活性，我们的测定法定义了一种新的工具来研究对细胞/生物反应给出化合物（S）的作用。例如，使用在不存在"目标假说"，它导致了160显示免疫调节活性的潜在的命中鉴定4398化合物进行初筛。因此，使用该测定的是适合用于药物开发的程序探索进一步在体外 /体内验证研究之前大化学文库。

高温超导实验的设计设计此荧光分析时有两个重要的因素考虑在内。首先，我们需要复制在其中的T或B细胞激活将代表一个疾病（ 例如移植物抗宿主病）的生理状态。第二，应该使用灵敏和定量的方法来进行细胞活化的评估。荧光是时下高温超导读数的主要选择之一，因为它符合这些需求。 ...

Watch this Scientific Journal Video about A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules at JoVE.com

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

我们提出在目前的研究使用从转基因小鼠衍生的淋巴细胞的新颖的基于荧光的测定法。该测定是适于赋予的任抑制或促进淋巴细胞的活化的能力的小分子的高通量筛选（HTS）。

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 ...

a-fluorescence-based-lymphocyte-assay-suitable-for-high-throughput

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.3K 次观看.  Université de Montréal. 我们提出在目前的研究使用从转基因小鼠衍生的淋巴细胞的新颖的基于荧光的测定法。该测定是适于赋予的任抑制或促进淋巴细胞的活化的能力的小分子的高通量筛选（HTS）。 

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

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  ...

科研

医学

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

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

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

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

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

免疫与感染

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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 ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

化学抑制剂库筛选 t 细胞受体信号的中介识别

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

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

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 ...

<meta charset="utf8"></head><body>使用双报告基因的 脓肿分枝 杆菌高通量筛选测定

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>作者聚焦:用于高效发现 脓肿分枝杆菌 治疗的可扩展药物筛选方案

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

分枝杆菌高通量药物筛选的分析开发

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

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使用自制双辉光萤光素酶检测的高通量筛选功能

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分枝杆菌高通量药物筛选的分析开发

分枝杆菌高通量药物筛选的分析开发