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>

<ol>
	<li>Swinney, D. C., Anthony, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+were+new+medicines+discovered.">How were new medicines discovered.</a> Nat Rev Drug Discov. 10 (7), 507-519 (2011).</li><li>Macarron, R., 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=Impact+of+high-throughput+screening+in+biomedical+research.">Impact of high-throughput screening in biomedical research.</a> Nat Rev Drug Discov. 10 (3), 188-195 (2011).</li><li>Lokey, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+chemical+genetics:+progress+and+obstacles+on+the+path+to+a+new+pharmacopoeia.">Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia.</a> Curr Opin Chem Biol. 7 (1), 91-96 (2003).</li><li>Hall, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoproteomics-driven+drug+discovery:+addressing+high+attrition+rates.">Chemoproteomics-driven drug discovery: addressing high attrition rates.</a> Drug Discov Today. 11 (11-12), 495-502 (2006).</li><li>Pruss, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+strategies+for+neurodegenerative+diseases:+a+pathway+to+discover+novel+drug+candidates+and+potential+disease+targets+or+mechanisms.">Phenotypic screening strategies for neurodegenerative diseases: a pathway to discover novel drug candidates and potential disease targets or mechanisms.</a> CNS Neurol Disord Drug Targets. 9 (6), 693-700 (2010).</li><li>St Onge, R., Schlecht, U., Scharfe, C., Evangelista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+chemical+genetics+in+yeast+for+discovery+of+chemical+probes+targeting+metabolism.">Forward chemical genetics in yeast for discovery of chemical probes targeting metabolism.</a> Molecules. 17 (11), 13098-13115 (2012).</li><li>Houston, J. G., Banks, M. N., Binnie, A., Brenner, S., O'Connell, J., Petrillo, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Case+study:+impact+of+technology+investment+on+lead+discovery+at+Bristol-Myers+Squibb.">Case study: impact of technology investment on lead discovery at Bristol-Myers Squibb.</a> Drug Discov Today. 13 (1-2), 44-51 (2008).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4 (2), 67-73 (1999).</li><li>Walters, W. P., Namchuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+screens:+how+to+make+your+hits+a+hit.">Designing screens: how to make your hits a hit.</a> Nat Rev Drug Discov. 2 (4), 259-266 (2003).</li><li>Zheng, W., Thorne, N., McKew, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screens+as+a+renewed+approach+for+drug+discovery.">Phenotypic screens as a renewed approach for drug discovery.</a> Drug Discov Today. 18 (21-22), 1067-1073 (2013).</li><li>Malo, N., Hanley, J. A., Cerquozzi, S., Pelletier, J., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+practice+in+high-throughput+screening+data+analysis.">Statistical practice in high-throughput screening data analysis.</a> Nat Biotechnol. 24 (2), 167-175 (2006).</li><li>Moran, A. E., 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=T+cell+receptor+signal+strength+in+Treg+and+iNKT+cell+development+demonstrated+by+a+novel+fluorescent+reporter+mouse.">T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse.</a> J Exp Med. 208 (6), 1279-1289 (2011).</li><li>Kovacic, N., M&#252;thing, J., Marusic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistological+and+Flow+Cytometric+Analysis+of+Glycosphingolipid+Expression+in+Mouse+Lymphoid+Tissues.">Immunohistological and Flow Cytometric Analysis of Glycosphingolipid Expression in Mouse Lymphoid Tissues.</a> J Histochem Cytochem. 48, 1677-1689 (2000).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+estimation+of+DNA+and+RNA+content+in+intact+cells+stained+with+hoechst+33342+and+pyronin+Y.">Flow cytometric estimation of DNA and RNA content in intact cells stained with hoechst 33342 and pyronin Y.</a> Cytometry. 2, 143-150 (1981).</li><li>Zanella, F., Lorens, J. B., Link, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+screening:+seeing+is+believing.">High content screening: seeing is believing.</a> Trends Biotechnol. 28 (5), 237-245 (2010).</li><li>An, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+assays.">Fluorescence-based assays.</a> Methods Mol Biol. 486, 97-107 (2009).</li><li>Kishimoto, H., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+TCR+ligation+without+costimulation+causes+rapid+onset+of+Fas-dependent+apoptosis+of+naive+murine+CD4++T+cells.">Strong TCR ligation without costimulation causes rapid onset of Fas-dependent apoptosis of naive murine CD4+ T cells.</a> J Immunol. 163 (4), 1817-1826 (1999).</li><li>Schluns, K. S., Kieper, W. C., Jameson, S. C., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-7+mediates+the+homeostasis+of+naive+and+memory+CD8+T+cells+in+vivo.">Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo.</a> Nat Immunol. 1 (5), 426-432 (2000).</li><li>Jiang, Q., 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=Cell+biology+of+IL-7,+a+key+lymphotrophin.">Cell biology of IL-7, a key lymphotrophin.</a> Cytokine Growth Factor Rev. 16 (4-5), 513-533 (2005).</li><li>Koenen, P., 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=Mutually+exclusive+regulation+of+T+cell+survival+by+IL-7R+and+antigen+receptor-induced+signals.">Mutually exclusive regulation of T cell survival by IL-7R and antigen receptor-induced signals.</a> Nat Commun. 4, 1735 (2013).</li><li>Vella, A., Teague, T. K., Ihle, J., Kappler, J., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+4+(IL-4)+or+IL-7+prevents+the+death+of+resting+T+cells:+stat6+is+probably+not+required+for+the+effect+of+IL-4.">Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4.</a> J Exp Med. 186 (2), 325-330 (1997).</li><li>Hawkins, C. J., Vaux, 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=The+role+of+the+Bcl-2+family+of+apoptosis+regulatory+proteins+in+the+immune+system.">The role of the Bcl-2 family of apoptosis regulatory proteins in the immune system.</a> Semin Immunol. 9 (1), 25-33 (1997).</li><li>Sumantran, V. 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，由于其繁琐的要求和低灵敏度。 23虽然发光已经被提出作为一种替代比色测定法，这些发光测试中的HTS应用通常仅限由于细胞裂解的要求。尽管其他非裂解的协议进行了脱开发出来的生物发光信号的强度可以由许多因素，如萤光素吸收，辅因子，pH值可用性和培养基或缓冲液的透明度受到阻碍，从而导致检测到的生物发光信号，并在细胞活性的实际变化之间的差异。 24因此，大多数测定依赖于荧光的方法。事实上，基于荧光的测定法是用小型，高荧光有机能够监视多种在细胞活化细胞过程的分子最初开发。总而言之，荧光测定法具有更高的灵敏度，并且可以HTS期间可以容易地适应用于表型测量。 23 这里介绍的开发了基于荧光检测HTS有三大优势。首先，它是高度可靠的，可重复使用同一stimula已采用能够调节两种不同的初级细胞的化合物（T和B淋巴细胞）的筛选灰/读出的概念。例如，4398的化合物的初步筛选，这是完成了五个不同的运行显示了一致活化诱导的GFP以最小的内和测定间变异（&lt;5％分别为T细胞测定和&lt;15％，）信号。其次，测定使用市售的转基因小鼠模型。通常，基于荧光的测定法的设计需要主细胞的基因工程或感兴趣，以便显示在刺激时的荧光信号的细胞系。虽然这种方法生成一个宝贵的实验室工具，它一般不容易获得科学界。绕过此限制，T细胞和B细胞从Nur77 GFP转基因小鼠中分离。这些淋巴细胞的优点是它们对表达在T细胞受体或BCR刺激24小时内的GFP的能力。因此，筛选药物能调节转录机械驱动GFP表达还是整体移动的响应表示吸引力的方法。三，补充T细胞在其初始激活阶段CD3 / CD28珠要求除去之前GFP评估这些珠子。如果用于分析T细胞活化/抑制作为离去珠粒否则会掩盖GFP信号显微镜，这是很重要的。如果流式细胞仪来代替用于分析，珠子能在孔中留下（除非特别创建附加混杂限制（多个）到测定），为感兴趣的特定群体可以同时使用前向和侧向散射被选通。 最初的目标是设计用最小的细胞处理筛选试验。根据这样的背景下，T或用未分级的脾B细胞的活化刺激的珠或分别被限制淋巴细胞的很大比例的可溶性抗体/重组CD40L保持GFP阴性（ 图1）。作为此步骤是为成功的关键测定的，该协议被修改，以纯化使用市售的试剂盒，以增加的比例的GFP表达细胞T细胞或B细胞。这种方法显著改善两种细胞群的激活结果。虽然所选择的另一种方法，可以如有必要可用于T细胞和B细胞的分离，该方法是:ⅰ）高度重现，ii）容易适于通过流式细胞HTS之前评估隔离种群的纯度，ⅲ）和时间高效的纯化步骤是相对短的（约30分钟），并能以最小的制剂来进行。然而，必须指出的是，在本研究中提出的测定法是基于使用小鼠原代细胞。仲（确认的目的）和叔测定（在感兴趣的目标最终验证）应当按照主屏幕，以验证所确定的匹配进行。虽然也可以很容易地适应各种不同的细胞系中，i的药理作用dentified命中需要在其它物种（ 例如人类细胞-例如叔测定的）进一步验证，因为没有可能的方式来预测物种间持续的影响。总之，该测定是为识别的新的潜在的命中和/或分子的目标与从体外 /动物试验对人的结果的可能外推有价值。 即使设计测定法可以利用为T和B细胞，一个优点是赋予在这种情况下的具体使用的T细胞的。 TCR活化通常需要通过主（ 例如抗CD3或抗TCR）赋予双重刺激和二次共活化（ 例如抗CD28）的信号。 25， 第26有趣的是，抗CD3和抗CD28抗体可购买直接联系到的磁性珠的表面，并可以从细胞悬浮液中加入t前，使用标准磁体去除他化学化合物在筛选测试。为用于与未知的化学实体库，保留激活自由结合到其TCR复合物可以最小化试验化合物和那些结合于TCR结合槽之间的任何可能的干扰分子的T细胞。 27此外，除去刺激剂下面的T细胞的活化模拟生理情况，而不是与经受持续超生理TCR活化的T细胞的工作。后一种情况可能是有问题的，特别是如果它是在低浓度的筛选中使用它可能覆盖一个给定化合物的药理作用。因为有施加于B细胞具有相同的效果没有市售珠，这将是很有意思的，将来的测试执行HTS刺激的B细胞上的连续呈现是否限制了测定的性能。

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 border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:211px;">Nur77GFP mice</td>
			<td style="width:157px;">The Jackson Laboratory</td>
			<td style="width:135px;">Mouse strain No. 016617</td>
			<td style="width:224px;">An in house colony was established at our animal facility&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">96 wells-U culture plates, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">10062-902</td>
			<td style="width:224px;">T-cell activation using the magnetic beads</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">70&#181;m cell strainer, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352350</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:211px;">5 ml polystyrene round bottom tubes, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352058</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:211px;">50 ml polypropylene conical bottom tubes, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">89039-656&#160;</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">10 ml syringe without needle, sterile</td>
			<td style="width:157px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305482</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">T-25 culture flask</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">690 175</td>
			<td style="width:224px;">To incudabte B cells during activation</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">5 ml cell culture dish, sterile</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">627 160</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Penicillin- Streptomycin (10,000 U/mL)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">450-200-EL&#160;&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">RPMI 1600 with sodium bicarbonate and L- glutamine</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">350-002-CL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:211px;">MEM non-essential amino acids</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">321-010-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HEPES free acid 1 M</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">330-050-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Sodium pyruvate solution (100mM)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">600-110-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:211px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">080-910&#160;</td>
			<td style="width:224px;">Component of the splenocyte media and flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Phosphate buffered saline (PBS)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">311-010-CL</td>
			<td style="width:224px;">Component of flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">2-Mercaptoethanol (55mM)</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">21985-023</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:211px;">T- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19851</td>
			<td style="width:224px;">To isolate T cells</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:211px;">B- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19854</td>
			<td style="width:224px;">To isolate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Mouse T-Activator CD3/CD28 superparamagnetic beads</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11452D</td>
			<td style="width:224px;">To activate T cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Cell isolation magnet</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">18000</td>
			<td style="width:224px;">To isolate T cells and remove the magnetic beads&#160;</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG + IgM (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:135px;">115-006-068</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Recombinant Murine IL-7</td>
			<td style="width:157px;">Peprotech</td>
			<td style="width:135px;">217-17&#160;</td>
			<td style="width:224px;">To support T-cell survival during activation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Recombinant CD40L</td>
			<td style="width:157px;">R&#38;D Systems</td>
			<td style="width:135px;">8230-CL/CF</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD3 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">561799</td>
			<td style="width:224px;">To stain T cells for flow-cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD19 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">553786</td>
			<td style="width:224px;">To stain B cells for flow-cytometry</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:211px;">Biomed FXp</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">A31842</td>
			<td style="width:224px;">To re-suspend cells after 24 hours incubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Opera Phenix High Content Screening System</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">HH14000000</td>
			<td style="width:224px;">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。 10，16 作为可以得到以下Nur77 GFP一个健壮的GFP读出在体外 或体内衍生T细胞或B细胞的刺激，12我们的测定法利用此战略作为第i的工作模型非活化和活化的细胞之间进行区分免疫调节化合物的dentification。 最初，我们的测定法的设计使用未分级刺激的脾细胞为基础的GFP评估通过流式细胞仪。在这种情况下，既非活化/活化的T和B细胞用抗CD3或抗CD19，GFP评估分别之前。如在图1A中所示，T细胞（CD3 +细胞）表示一个给定的小鼠脾脏的大约30％。在稳定状态下，将GFP水平在10％的范围内（最有可能是由于在周边发展或稳态刺激期间通过TCR后胸腺选择）。以下使用的CD3 / CD28珠粒TCR刺激，在CD3 + T细胞中检测到绿色荧光蛋白表达的百分比五到六倍的增加（57％而不是10％）（ 图1A - B）。同样地，B细胞（CD19 +细胞）占总脾细胞的50-55％和其基底的GFP前PRESSION比得上未活化的T细胞（ 例如 ，〜10％， 图1C）。有趣的是，然而，使用抗小鼠IgG / IgM和重组CD40L的BCR刺激引发GFP表达一个微不足道的增加（33％，而不是12％， 图1C - D）。如GFP表达为评估对活化的T细胞或B细胞中筛选的小分子的药理学效果的关键要素，优化其强度是中央为筛选的灵敏度和成功。 使用纯化的T细胞或B细胞的GFP表达的评估虽然T和B细胞的刺激可以直接使用未分级的脾细胞来实现，所述GFP表达强度不是用于筛选足够敏感，特别是如果读数是通过显微镜来进行。为了提高细胞应答，T细胞，首先通过磁纯化使用市售的试剂盒（ 图2A）珠。如先前使用未分级的脾细胞中所示，分离的CD3 + T细胞的10％为GFP阳性（基础表达）。在这种情况下，使用CD3 / CD28珠粒T细胞刺激显著改善了他们的GFP响应（ 图2A - 2B，相对于57％时，使用普通的脾细胞86％）。与B细胞（ - 2D，80％，而不是33％。图2C）获得了类似的改进。这些结果清楚地证明，使用纯化的T细胞和B细胞的最大化GFP表达，这是适合于所提出的表型筛选。 所设计的HTS试验示意图总体而言，测定可以被细分为四个主要部分（ 图3）。在T细胞为例子的情况下，一个脾细胞细胞悬浮液，以提纯T细胞（步骤1-3），接着用CD3 / CD28珠粒体外一个12小时的刺激步骤制备（步骤4-5）。然后活化的T细胞铺板并在384孔板使用自动HCS系统GFP和Hoechst的强度的评估之前选择的小分子（步骤6-7）24小时进行培养。相同的测定可用于B细胞与在步骤4-5进行的修改。简言之，B细胞可以与抗小鼠IgG / IgM和重组CD40L而不是珠刺激。由于这些部件在它们的可溶形式使用（ 例如不挂珠），B细胞可以被培养12小时，然后简单地在用于筛选过程384孔板电镀之前洗涤。 高效的T细胞存活是原始的HTS研究的成功。因此，两个限制因素可能妨碍测定的质量，并应考虑。第一鼠T细胞是下列体外刺激高度敏感的细胞凋亡。 17第二，所有测试的化合物稀释在DMSO溶液，这可能会增加第二压力因子。为了最大限度地减少细胞的损失，重组IL-7（2毫微克/毫升）的混合物在步骤4-5在TCR刺激补充到T细胞。这种稳态的细胞因子支持增殖和通过抗凋亡分子如Bcl-2，Bcl-xL的和Mcl-1的表达增强了T细胞的存活。 18，19，20，21，22 的4398化合物主要高通量筛选揭示了T细胞活化的几个活化剂和抑制剂高通量筛选是通过电镀的非激活（负共75000进行ntrol）或显示在图4中的活化T细胞（阳性对照）。以保证高再现性，该测定用0.87（ 图5A）的所获得的Z参数进行数次。使用HCS系统，刺激T细胞的观察GFP的表达比未刺激的T细胞，允许区"一""的操作窗口"，其中可以容易地检测的GFP的抑制性化合物（ 图5A）的产生高20倍。这显示在该图中由细胞的总数中的扫描场划分荧光细胞的数目方面。在图5A所示的例子中，320的化合物的筛选推出三种化合物（指出出由红色箭头），它能够通过1.5-2.5倍抑制GFP表达的。在本实施例中，阳性对照值为0.44±0.02和阴性对照为0.02±0.00。 钍共有4398化合物（0.85 Z参数）电子筛选，然后进行。由药物化学家作为代表含136000实体（ 图5B）的整体化学品库根据其化学结构种子/代表性化合物进行了选择的化合物。在测定结束时，GFP抑制的分析，鉴定能够既抑制（正转向块）或增强的GFP表达（负转向块）的化合物。 <img alt="图1" src="/files/ftp_upload/55199/55199fig1.jpg" /> 图1:以下TCR或普通的脾细胞进行的BCR活化T-分析和B细胞的GFP表达。 （A）与抗CD3使用脾细胞在不存在（左上面板）的CD3 / CD28珠存在（右上面板）抗体或染色的T细胞的代表性流式细胞仪分析。这两个T细胞群GFP表达显示在底部。由T细胞的流式细胞仪分析得到的GFP表达的（B）中所占的百分比。误差线代表平均值±标准差。 （C）与（A）中的类似，只是脾细胞使用抗小鼠IgG / IgM抗体激活和重组CD40L然后染色的CD19。由B细胞的流式细胞仪分析得到的GFP表达的（D）的百分比。误差线代表平均值±标准差。所有所示实验至少重复3次（n = 5 /组; * P &lt;0.05和*** P &lt;0.001）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55199/55199fig1large.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/55199/55199fig2.jpg" /> 图2:使用以下分别TCR或BCR活化纯化的T细胞或B细胞GFP表达分析。 （A）Repres用抗CD3抗体染色的纯化的T细胞的entative流式细胞仪分析。的GFP诱导，纯化的T细胞，用CD3 / CD28珠粒（右上面板）刺激。未活化的对照T细胞在左上方面板被示出。两个未活化和活化的T细胞的GFP表达在底部被显示。由T细胞的流式细胞仪分析得到的GFP表达的（B）中所占的百分比。误差线代表平均值±标准差。 （C）与（A）中的类似，只是B细胞的纯度通过CD19染色评估，并用抗小鼠IgG / IgM和重组CD40L激活。由B细胞的流式细胞仪分析得到的GFP表达的（D）的百分比。误差线代表平均值±标准差。所有所示实验至少重复3次（n = 5 /组和*** P &lt;0.001）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55199/55199fig2large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <img alt="图3" src="/files/ftp_upload/55199/55199fig3.jpg" /> 图3:表示表型筛选的步骤总体示意图。有准备HTS检测所需的7大步骤。一位女Nur77 GFP鼠标牺牲分离其脾（步骤1）。以下一个脾细胞悬浮液（步骤2）的产生，T细胞是负（由不想要的细胞的耗竭）使用市售的试剂盒（步骤3）中分离。分离的T细胞，然后用重组IL-7在37℃（步骤4）不存在或CD3 / CD28珠存在下，在圆底96孔板温育。 12小时后（第5步）中，T细胞被吸几次以去除使用原本用于T细胞纯化相同的细胞分离磁体之前珠粒去除任何聚集体。分离的T细胞，然后在384孔板（75 000 CEL接种LS /孔，步骤6）用另外24小时（步骤7）的化合物。次日，孵育细胞用Hoechst 30分钟由HCS系统（步骤8）分析前染色。在相同的设置可以是随访基于小区B测定的制备除了使用步骤4和5是通过简单的洗涤来代替作为刺激剂在可溶形式加入。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55199/55199fig3large.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/55199/55199fig4.jpg" /> 图4:布局T细胞在384-孔板的电镀。在测定中使用的每个板包含三组。在第一组中，孔装载有非活化的T细胞（为在板的各侧面的GFP阴性对照;孔A1-P1和A24-P24），而第二组包含活化的T细胞无DRUGS（GFP的阳性对照;井A2-P2和A23-P23）。含有激活的所有剩余孔补充有在筛选中使用的化学实体的T细胞。所有孔中含有0.5％的DMSO。对于GFP和Hoechst的信号的例子示为未活化和活化的T细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55199/55199fig4large.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/55199/55199fig5.jpg" /> 图5: 主表型筛选 。 （A）320化合物进行了初步筛选的代表性的例子。非活化的T细胞是相反的TCR刺激的T细胞，从而显示器20倍高的GFP表达几乎GFP空。 （B）的所有化合物的累积数据中的测定法进行筛选。该GFP表达证实化合物迪张开抑制作用，而其它化合物，分别表现为T细胞的刺激。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55199/55199fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

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

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

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

定量高通量单细胞的细胞毒性T细胞的测定

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

科研

免疫与感染

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

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

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

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

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

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

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

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

高通量筛选RNA病毒的广谱化学抑制剂

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

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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

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

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

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

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

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...