Name: establishment and optimization of a high throughput setup to study staphylococcus epidermidis and mycobacterium marinum infection as a model for drug discovery
Uploaded: 2014-06-26T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discover

我们感谢莱奥妮·德波尔和BAS Zaat（学术医学中心），为我们提供了S。表皮 O型47株。我们感谢波多黎各邦加茨，弗朗西斯迪斯和Angela昏迷（联盟Biometrica）寻求帮助和建议的COPAS XL和广阔的生物影像分析仪分析。我们感谢戴维德威特，乌尔里克Nehrdich和劳拉面包车许尔斯特鱼照料，并从莱顿大学的其他同事的有益讨论。本研究形成了生物医用材料研究所共同资助由经济事务荷兰财政部的研究计划的项目P5.03 IBIZA的一部分，和经济事务的荷兰部的智能混合编程（NWOA_6QY9BM）和荷兰教育，文化和科学。从欧盟项目ZF-健康（FP7-健康-2009-242048），获得额外的支持，以及RMJ是由居里夫人奖学金作为欧盟首次培训网络FishForPharma（PITN-GA-201有经验的研究员支持1-289209）。资源，其中SJR收到的资金从创新药物行动联合承诺项下出让协议编号为115337，是由来自欧盟第七框架计划（FP7/2007-2013）和EFPIA公司以实物contribution.The作者进一步确认捐款从莱顿大学基金（LUF）用于机器人和从Cyttron的财政支持，在Besluit补贴Investeringen Kennisinfrastructuur程序，而这又是经济由荷兰科学研究组织为成像设备的支持。该COPAS系统采集部分由来自荷兰科学研究组织（NWO，834.10.004）的财政援助为地球和生命科学（ALW）司的支持。

1，细菌菌株和生长条件<ol><li>准备S。表皮接种<ol><li>需要几个单个菌落从S。表皮葡萄球菌菌株O-47，含有pWVW189从卢瑞亚BERTANI（LB）琼脂平板上于25毫升的LB培养基中补充有10微克/毫升氯霉素和培养过夜，在37℃下补充有派生的mCherry表达载体（德波尔L.未发表） 10微克/毫升氯霉素至对数中期阶段。 </li><li>离心机将1ml培养物在12,000 xg离心1分钟，随后冲洗，3倍，用1ml的无菌磷酸盐缓冲盐水（PBS）与0.3％（V / V）吐温80。 </li><li>测量的光密度在600nm（OD 600），和稀的细菌悬浮液至OD 600为0.3的2％（重量/体积）聚乙烯吡咯烷酮40（PVP 40）在PBS中。注:OD 600为0.3对应于1.0×10 8菌落形成单位/毫升（CFU /毫升）。 </li></ol&gt; </li><li>准备M.海鱼接种<ol><li>需要几个单个菌落从M。海鱼株M或E11包含pSMT3-mCherry的载体稳定表达的mCherry 10从米德尔7H10琼脂平板上，10％（V / V）米德尔OADC富集补充50微克/毫升，在28℃下在10毫升潮霉素和文化过夜的Middlebrook 7H9肉汤中，用10％（V / V）的Middlebrook ADC富集补充有50微克/毫升潮霉素。 </li><li>离心机将1ml培养物在12,000 xg离心1分钟，随后洗涤用1毫升无菌PBS中与0.3％（V / V）吐温80 3倍。 </li><li>测定OD 600，并稀释细菌悬浮液至OD 600为0.3的2％（重量/体积）的PVP 40的PBS中。注:OD 600为1对应于1.0×10 8 CFU /毫升。 </li></ol></li></ol> 2，准备斑马鱼鸡蛋<ol><li>将最多70男和50女百搭TYPE分别为斑马鱼的大型养殖船。注意:在大容器繁殖的下部放置在雌性鱼。 </li><li>在早晨除去隔板的第二天，让斑马鱼开始繁殖。 </li><li>通过卵收集器在一个50毫升管填充有蛋水（60μg/ ml的瞬间海洋海盐）收集鸡蛋在大型饲养容器的底部。 </li></ol> 3，注射针头<ol><li>获得市售定制玻璃毛细管针与10μm的内径。 </li></ol>注射4。实验大纲<ol><li>熬百毫升1％（W / V）琼脂糖鸡蛋水，凉，直到大约40ºC。倒入琼脂糖进入自动microinjectors板和放置1,024井邮票到琼脂糖。注:该板块已准备就绪，当冷却使用。 </li><li>在自动化的微量操作软件，点击"校准阶段&#39;，然后点击&#39;1024&#39;以及电网和地方琼脂糖板块中微量和通过点击屏幕上的井中心位置校准板。 </li><li>转到&#39;针菜单"，然后点击"校准持针器"。 </li><li>使用微加载尖含100 CFU / NL S。无论是10微升的pvp 40填补了注射针表皮或30 CFU / NL M。海鱼 ，或使用PVP 40为模拟注射。 </li><li>将针在自动微喷射器和通过降低或移动针上并点击屏幕上的针的位置校准的x，y位置。然后通过点击屏幕上的针的前端的位置校准针的z轴位置。 </li><li>分发鸡蛋在使用塑料移液管琼脂糖网格，并去除多余的蛋水。将琼脂糖电网自动化的微量。 </li><li>转到"注射液n菜单"和调整"射出压力"设置为200百帕，&#39;注射时间"0.2秒，补偿压力"15百帕，这与1 NL，在Femtojet设置菜单。 </li><li>点击"全部注入&#39;到整个板块注入。 </li><li>收集鸡蛋注入洗成一个培养皿（92 x 16毫米），最大每个培养皿7​​0胚胎和孵化，在28°C。后</li></ol> 5，流式细胞仪分析<ol><li>制备大颗粒流，根据制造商的指示仪，并填写样品杯和鞘液容器，蛋水。 </li><li>在操作软件，进入"PMT"菜单，并使用以下设置:650 V为&#39;红&#39;通道和0 V为&#39;绿色&#39;和&#39;黄色&#39;的渠道。然后去"阈值"菜单，并使用以下设置:&#39;光学升密度&#39;阈值信号:975毫伏（COPAS XL值:50）和"行程时间"（TOF）最低320微秒（COPAS XL值:800），以减少碎片的影响。 </li><li>用于分析，而不必排序胚转至步骤5.4中，对于分析和分选的胚胎到培养皿转到步骤5.5或用于分析和分选的胚胎成96孔板转到步骤5.6。 </li><li>放置在样品杯的胚胎，然后点击"开始"开始分析。当所有的胚胎进行了分析停止分析通过点击"停止"。通过单击"将所有&#39;保存数据。注:所有数据都为TXT，LMD，DAT，CSV和BSRT文件存储。按照协议，在步骤5.7。 </li><li>将胚胎在样品杯中，并确定每板70胚的最大将由在"排序"菜单中输入70来分类的。将空的培养皿分拣机下，点击"手动排序"。当彼得二<html> SH被填满后，通过点击"将所有&#39;保存数据。注:所有数据都存储为TXT，LMD，DAT，CSV和BSRT文件。按照协议，在步骤5.7。 </li><li>将胚胎在样品杯中，并确定每孔1胚胎的最大将由在"排序"菜单中输入1来分类的。将空的96孔板到左侧板固定器，并单击"填充板"。当96孔板被填满后，通过点击"将所有&#39;保存数据。注:所有数据都存储为TXT，LMD，DAT，CSV和BSRT文件。按照协议，在步骤5.7。 </li><li>得到TXT文件来处理原始数据，使用下面的数据过滤器设置:"状态选择":40，如果使用排序模块的状态排序":6然后从"红使用数字从总的荧光信号"信道来计算平均值和平均值的标准误差。绘制这些数据集到酒吧或散点图。 </li></ol>ve_title"&gt; 6。药物治疗<ol><li>分析和排序在后3天注射剂（DPI），M。使用大颗粒流动细胞仪（步骤5.5） 海鱼感染胚胎在两个相等的组。治疗一组与兴趣在其载体溶剂中的化合物等与载体溶剂（对照）。采用类似的处理嘲笑注射的对照测试抗生素的副作用。 </li><li>重复第4和第5 DPI分析（步骤5.5）和刷新蛋水或含有该化合物的蛋水。 </li></ol> 7，高分辨率成像<ol><li>麻醉的胚胎用0.02％（重量/体积）在蛋水缓冲3 - 氨基苯甲酸乙酯（三卡因）10分钟，在分析前。 </li><li>根据制造商的说明制备脊椎动物自动筛分技术系统和大粒子采样。 </li><li>选择从"影像对应于胚胎的年龄的参考图像 - 物体211;检测设置"菜单。 </li><li>选择的照片被从"成像 - 自动存储图像&#39;所作的脊椎动物自动筛分技术系统的量和方向的菜单。 </li><li>将96孔板充满了胚胎（步骤5.6）到大颗粒采样器的左侧板夹，并单击"运行板"。 </li><li>当胚胎被检测到并正确地定位;像头和单独使用的激光共聚焦显微镜10倍平原干燥目的，并使用图像处理软件拼接后图像的尾巴。 </li></ol>

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JS是生命科学的方法BV公司老板产生这篇文章中所使用的工具。

在本文中描述的高通量方法提供了一种快速和成本有效的管道来筛选大量的鱼的胚胎和幼虫的不同类型的感染。使用大的繁殖容器，而不是传统的单人或家族繁殖坦克容易控制的产卵过程和产生较大的同步蛋的数量。随着自动化微注射系统7的改进版本，它可以在相同的细胞阶段在1小时内注入高达2500鸡蛋几乎所有。有了这些更新和改进的软件是可行的注入更多的鸡蛋比以前可能是它可以用来与细菌增殖的读出进行大量药物屏幕。然而，这种方法目前还仅限于蛋黄注射液，由贝纳尔等人描述，例如其他注射途径。 （2012）6，将可望在自动化微注射系统结合在不久的将来。 &lt;P类="j​​ove_content"&gt;虽然这些方法为基准，筛选斑马鱼，它会与其它鱼种的应用程序很有用。例如，常见的鲤鱼已表明具有的优点为药物屏幕。像斑马鱼，鸡蛋和鲤鱼早期胚胎是透明的，但随着几百几千只蛋和自交系，提供更稳定的遗传背景16的可用性它的大尺寸产卵的主要优势。 大量受感染的胚胎的分析是通过对高吞吐量大颗粒流式细胞仪。这种装置可以排序分析胚胎成多孔板或培养皿中使其特别适于测试大量化合物。如果需要更高的成像分辨率，比设置适合的方式，大颗粒的流式细胞仪技术可用于预筛分，并随后通过分析样品在一个中等摆在更高的分辨率。这可以使用脊椎动物自动筛分技术17，18来完成。该设备可以自动收集从多孔板或散装容器，像360°通过利用激光共聚焦显微镜或立体显微镜毛细管2和7天受精后的活的或固定的胚胎和2散装容器允许胚胎基于人工分拣再处理在显微图像。未来的改进将使得胚胎的成像到多孔板后的排序，因此使得有可能用CLSM屏幕自动大量单个胚胎的一段时间。假设在将来的应用程序的脊椎动物自动筛分技术系统也可以连接到大颗粒的流式细胞仪技术，无需事先分配幼虫的需要为多孔板，将导致一个更先进的分拣。 本文详细介绍了建立高吞吐量的设置，研究S的ð优化表皮和M.海鱼感染为模型药物发现。它表明，这些细菌注射到卵黄的结果依赖于卵在注射时的发育阶段。注入M.海鱼 E11在16-128细胞阶段或M个菌株在16-64细胞阶段导致相同的感染图案作为尾静脉注射2，6。但是这种设置并不限于只细菌病原体的增殖。有可能分别为13至机器人注入含有DNA，RNA或吗啉为转基因溶液，过表达和基因敲除的研究中，它是前图所示。另外，它表明，这种设置也用于癌症细胞增殖和迁移的研究是有用的。因此，这条管道呈现为多种信号机制的高通量筛选的背景下，一个通用的方法先天免疫的，应用于感染性疾病和癌症的发展。这些屏幕可以与他人进行药物发现也与确定适用的药物可能的毒性效应分析相结合。

迄今为止，斑马鱼（ 斑马鱼 ）已经成功地建立，以研究各种传染病1的有效模型。斑马鱼的胚胎提供了独特的体内成像的可能性，由于其透明度和大量表达荧光蛋白转基因存在记者行。这个强大的组合使得它可以及时跟踪不同的免疫细胞类型，同时与病原体如分枝杆菌，M的近亲互动结核2，或表皮葡萄球菌 ，生物材料相关感染3-5的主要病因。感染不同的路线可以在斑马鱼胚胎中使用根据研究6的目的。 其中一个感染途径是蛋黄注射的细菌。这种方法的主要优点相比其他的是蛋黄infecti上可以自动通过机器人注射来进行，显著减少喷射时间，并允许高的感染7,8的再现性。 以前的工作，利用斑马鱼作为对S的体内研究模型系统的高通量表皮和M.海鱼感染，证实是成功的7，8。该系统能够通过机器人蛋黄注射早期胚胎，并使用荧光读数用作细菌负荷的度量来筛查疾病的进展。在此概念的协议，这种设置得到了优化，并建立了高效高通量管道与产生大量均匀受感染的胚胎，并在与众多化合物治疗后的时间跟踪感染进展的可能性。既定的设定也能够产生高达8000同步胚胎筛选对于疾病进展，以这种方式达到每小时2500胚的处理。胚胎基于它们的细菌负荷分类的使用自动化系统，确保感染的幼虫同质群体。此外，为了验证设置，参考已知预防结核病的进展在哺乳动物的效果进行了测试感染分枝胚胎海鱼 E11株或毒性更强的M株9。 本文详细研究描述了已经成立，能够产生大量感染胚胎，并在发展和复合处理后的细菌进展的后续分析的高通量管道。

<table><tbody><tr><td>Middlebrook 7H10 agar</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>262710</td><td></td></tr><tr><td>Middlebrook 7H9 broth</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>271310</td><td></td></tr><tr><td>BBL Middlebrook oleic acid albumin dextrose catalase (OADC) enrichment</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>211886</td><td></td></tr><tr><td>BBL Middlebrook albumin dextrose catalase (ADC) enrichment</td><td>BD Bioscience, Franklin Lakes, New Jersey, USA</td><td>211887</td><td></td></tr><tr><td>LB agar</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>L5542</td><td>Multiple suppliers</td></tr><tr><td>LB broth</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>L3022</td><td>Multiple suppliers</td></tr><tr><td>Chloramphenicol</td><td>Bio-connect, Huissen, the Netherlands</td><td>16785.03</td><td>Multiple suppliers</td></tr><tr><td>Hygromycin</td><td>Bio-connect, Huissen, the Netherlands</td><td>25966.01</td><td>Multiple suppliers</td></tr><tr><td>Tween-80</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>P1754</td><td>Multiple suppliers</td></tr><tr><td>Polyvinylpyrrolidone40</td><td>Calbiochem, San Diego, California, USA</td><td>529504</td><td>Multiple suppliers</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich, Saint Louis, Missouri, USA</td><td>A5040</td><td></td></tr><tr><td>Agarose</td><td>Sphaero-Q, Gorinchem, the Netherlands</td><td>S103</td><td>Multiple suppliers</td></tr><tr><td>Instant ocean sea salt</td><td>Sera Marin, Heinsberg, Germany</td><td>5460</td><td></td></tr><tr><td>iSPAWN</td><td>Techniplast, Buguggiate, Italy</td><td>iSPAWN</td><td></td></tr><tr><td>Automated microinjection system</td><td>Life Science Methods BV, Leiden, the Netherlands</td><td>Automated microinjection system</td><td></td></tr><tr><td>Complex Object Particle Analyzer and Sorter XL (COPAS XL)</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>COPAS XL</td><td></td></tr><tr><td>Vertebrate Automated Screening Technology BioImager (VAST BioImager)</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>VAST BioImager</td><td></td></tr><tr><td>LP Sampler</td><td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td><td>LP Sampler</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica Microsystems, Wetzlar, Germany</td><td>TCS SL</td><td></td></tr><tr><td>Injection needle 10 &amp;micro;m inner diameter</td><td>Qvotek, Mississauga, Canada</td><td></td><td></td></tr></tbody></table>

establishment and optimization of a high throughput setup to study staphylococcus epidermidis and mycobacterium marinum infection as a model for drug discovery

斑马鱼正在成为药物发现放映临床前阶段的宝贵工具与高通量筛选的可能性整体动物模型。它们可以被用来弥补在早期阶段和基于细胞的测定法之间的间隙在体内验证在哺乳动物模型，减少，以这种方式，通过传递到测试的昂贵得多啮齿动物模型中化合物的数量。有鉴于此，本手稿中描述使用斑马鱼作为表皮葡萄球菌的研究和分枝杆菌感染的体内模型系统的新高吞吐量的管道。这种设置允许对大量同步胚的生成和分析均匀感染。此外，该管道的灵活性使用户可以容易地实现其他平台在需要的时候，以提高分析的分辨率。斑马鱼具有创新性的高吞吐量德在一起的结合chnologies打开，因为有大量的可用于破译的新化合物的作用方式提供的转基因株系的，不仅是因为使用的是整个动物模型，而且强度药物检测和发现新的可能性的领域。

本结果表明，该高通量管道来研究S。表皮和M.海鱼感染已经成功地建立，并且可以扩展到其他感染模型。首先，利用大型养殖容器（ 图1A），根据已公布的制度Adatto 等上。（2011）11，使产生大量的同步鸡蛋在单事件给予高度控制的产卵过程。下一个，以便能够在先前开发的自动化微注射系统7的改进版本被使用（ 图1A）在很短的时间期间注入大量的胚胎。以评估这是最好的发展阶段为卵黄感染，注射用S。表皮和M.海鱼分别在1和512细胞阶段之间的所有不同阶段中进行，根据金梅尔等人所作的描述（1995）12 </suP&gt;。 注射用100 CFU S。表皮 16和128细胞阶段之间提供了最好的感染图案（ 图2）。细菌增殖的蛋黄内3天，并扩散到身体3 dpi的开始。 16细胞阶段之前进行注射导致高死亡率从4 dpi时，并在注射后的256细胞阶段表现出主要是细菌生长的蛋黄内，几乎没有任何细菌的胚胎在体内扩散。通过荧光强度分析使用大颗粒流式细胞仪如由维尼曼等人进行的细菌负荷的定量。（2013）8（ 图3）。 观察结果表明，注射的30 cfu的分枝的最优发育阶段海鱼注射是16到128细胞阶段之间的E11菌株（ 图4A）和16-64之间细胞阶段的更致命M TG1的N（ 图4B）。胚胎在这些阶段中注入的显示细菌生长的蛋黄内，细菌通过胚胎（ 图7）扩散。感染两种菌株在早期阶段提出了广义的非特异性细菌生长导致胚胎后4 dpi的死亡。另一方面，在胚胎的后期注入细菌负荷被限制在蛋黄。 接着，预分类与大颗粒流式细胞仪（ 图1B）产生感染的鱼大的同质群体的扣除非或高度感染的胚胎（ 图5A和6A）。预分类后，M。海鱼感染胚胎分别用利福平，第一线抗结核药。以前的研究证明，用利福平治疗的200μM剂量有效地降低M.海鱼感染斑马鱼7，13。采取advantagE中的大量具有高吞吐量的设置，处理不同剂量进行产生均匀感染胚胎的。感染与M.胚胎海鱼 M株和48小时处理过的带12，24，和200μM利福平表明，以减少有效分枝杆菌感染中以剂量依赖方式（ 图5B）。鉴于有效减少使用利福平在200μM该浓度被用于以后的实验剂量的感染。与前面的结果行，细菌研究进展负担使用M。海鱼 E11菌株显著减少24小时，并用200μM的利福平治疗后开始观察到（ 图6B）。 此外，如果高倍率摄像需要这些胚胎的，它们可以被自动地在96孔板上（ 图1C），从那里的样本可以使用被分析显示脊椎动物自动筛选技术体系与大颗粒采样器安装到激光共聚焦显微镜。 脊椎动物自动筛分技术系统与大粒子采样是可以被安装在一个CLSM或立体显微镜的系统。该设备允许活的或固定的胚胎的装载从96孔板或散装容器自动地通过玻璃毛细管，并在所需的角度（ 例如 ，背侧或侧面）orientates它在照相 ​​机的前面。在所有方向胚胎的图像可以与板载相机或与外部激光共聚焦显微镜（ 图7）进行。胚胎随后将被转移在收集或废物容器。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig1highres.jpg" src="/files/ftp_upload/51649/51649fig1.jpg" /> 图1。主流实验OUTL国家统计局。大A）成鱼放在一起交配，鸡蛋收集，排列成琼脂糖片和注射B）注入卵孵化在28℃下，将预先分拣可能的药物治疗。 三）后续分析颗粒仪和/或大粒子采样/脊椎动物自动化技术筛选与流动激光共聚焦显微镜。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig1highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig2highres.jpg" src="/files/ftp_upload/51649/51649fig2.jpg" /> 图2。建立最佳小区级为S。表皮蛋黄注射。斑马鱼胚胎注射在蛋黄在不同的发育阶段，从1到512细胞阶段100 CFU的S.表皮 。胚胎1之间注入ND 8细胞阶段显示，从4 dpi的蛋黄和高死亡率的细菌生长。胚胎16和128细胞阶段之间注入表明在蛋黄和起始于3 dpi的身体内部的细菌生长。胚胎之间的256和512细胞阶段注入显示，蛋黄里面很多细菌的生长。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig3highres.jpg" src="/files/ftp_upload/51649/51649fig3.jpg" /> 图3，定量使用大颗粒流式细胞仪细菌的负担。100 CFU的S.表皮注射到斑马鱼胚胎的卵黄A）最多5 DPI，每一天，10个胚胎组均化并直接电镀，显示出基于两个生物副本（误差线的平均指数级增长= SEM）B）大颗粒的流式细胞仪分析显示了从非注入和S的平均荧光信号表皮注射胚胎。每个条件30-160胚胎进行了分析（误差棒= SEM），不同字母表示统计学显著差异通过单向ANOVA，然后进行Tukey的事后检验（P &lt;0.001），NS:菌落形成单位之间并没有显著差异C）的相关性10 S.组的平均荧光信号表皮葡萄球菌感染的胚胎（误差线= SEM）。这个数字已经被修改维尼曼等人 （2013年）8。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig4highres.jpg" src="/files/ftp_upload/51649/51649fig4.jpg" /> 图4。Establis最佳小区级分枝的hment 海鱼黄注射液。斑马鱼胚胎注射在所有不同的发展阶段，从1到512个细胞阶段，30 CFU M的海鱼 E11和M菌株A，B）从胚胎1-8细胞阶段注入的显示类似的扩展和间16-128细胞阶段的死亡率与两菌株A）胚胎注射E11菌株显示形成肉芽肿和全身感染而注入从256到512细胞阶段保持细菌负荷入蛋黄B）之间的胚胎16-64细胞阶段注入以M株显示形成肉芽肿样结构和全身感染而来自128注入512细胞阶段保持细菌负荷入蛋黄。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig4highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a> <img alt="图5" fo:内容宽度="5英寸"FO:SRC ="/ files/ftp_upload/51649/51649fig5highres.jpg"SRC ="/ files/ftp_upload/51649/51649fig5.jpg"/&gt; 图5。M.治疗海鱼急性感染与一线抗结核药物。胚胎16-64之间细胞阶段注入30 CFU M的海鱼 M株是通过在大颗粒上运行流式细胞仪在3 dpi的两组丢弃非和/或高度感染的胚胎。 甲）荧光单个胚胎的后两组进行排序B）胚胎与利福平（RIF处理）48小时在剂量12，24，和200μM在4 dpi的分析中;它们的细菌负荷显著降低。C）用DMSO和利福平在剂量12和24的处理的胚胎的代表性COPAS的档案和200μM的24小时。细菌负荷及分布由红峰表示。蓝线表示的元素来分类的（4档DPF斑马鱼Ëmbryo）由COPAS。每个条件60-90胚胎进行了分析。每个数据点代表一个单独的胚胎。值表示为平均值±标准差。 NS:没有显著差异。由单因素方差分析后Tukey的进行事后检验进行的差异显着性分析。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig6highres.jpg" src="/files/ftp_upload/51649/51649fig6.jpg" /> 图6。M.治疗海鱼慢性感染与第一线抗结核药物。胚胎16-64之间细胞阶段注入30 CFU M的海鱼 E11菌株通过大颗粒流在3 dpi的两组丢弃非和/或高度感染胚胎后进行排序运行流式细胞仪。<html> A）个别胚胎在两组荧光B）与利福平（RIF）在200微米〜4天处理的胚胎进行了分析显示1天的治疗后，一个显著减少细菌负荷。每个条件90胚胎进行了分析。值表示为平均值±标准差。不同字母表示同一处理时间点之间的显著差异。 *表示与对照组显著差异。 NS:没有显著差异。由单因素方差分析后Tukey的进行事后检验进行的差异显着性分析。 （P &lt;0.05）。图B）已经被修改Spaink 等人 （2013）13。 <a href="https://www.jove.com/files/ftp_upload/51649/51649fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> hres.jpg"SRC ="/ files/ftp_upload/51649/51649fig7.jpg"/&gt; M的图7。结果脊椎动物使用自动筛选技术和激光共聚焦显微镜海鱼 E11蛋黄注射成像。5 DPI FLI1-EGFP 14胚胎的共焦Z图库（缝合3图像）。M 的）现场胚胎细胞增殖表现海鱼 E11菌（红色）在整个身体。 二）固定5 DPI FLI-EGFP胚胎显示M。海鱼 E11细菌（红色）整个身体的共同定位与白细胞（淡蓝色）用L-网的免疫染色15检测。

Watch this Scientific Journal Video about Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discover

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

这个视频文章介绍了已成功建立感染和分析大量斑马鱼的胚胎提供了一个新的强大的工具化合物的检测和药物发现用整体动物脊椎动物有机体的高通量管道。

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

establishment-optimization-high-throughput-setup-to-study

Research

JoVE Journal

Immunology and Infection

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

13.1K 次观看.  Leiden University. 这个视频文章介绍了已成功建立感染和分析大量斑马鱼的胚胎提供了一个新的强大的工具化合物的检测和药物发现用整体动物脊椎动物有机体的高通量管道。 

视频: Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

科研

生物工程

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

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. ...

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 ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

<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. ...

建立和高通量安装程序研究优化

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此视频中的章节

芯片上的压力诱导抗生素易感性测试

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

高通量设置的建立筛选小分子能调节C-二GMP信令

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

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

微生物相互作用研究的高通量共培养测定

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

分枝杆菌高通量药物筛选的分析开发