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>

<ol>
	<li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+made+transparent+with+the+zebrafish+model.">Host-pathogen interactions made transparent with the zebrafish model.</a> Curr Drug Targets. 12, 1000-1017 (2011).</li><li>Tobin, D. M., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pathogenesis+of+Mycobacterium+marinum+and+Mycobacterium+tuberculosis.">Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis.</a> Cell Microbiol. 10, 1027-1039 (2008).</li><li>Boelens, J. J., 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=Biomaterial-associated+persistence+of+Staphylococcus+epidermidis+in+pericatheter+macrophages.">Biomaterial-associated persistence of Staphylococcus epidermidis in pericatheter macrophages.</a> J Infect Dis. 181, 1337-1349 (2000).</li><li>Broekhuizen, C. A., 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=Tissue+around+catheters+is+a+niche+for+bacteria+associated+with+medical+device+infection.">Tissue around catheters is a niche for bacteria associated with medical device infection.</a> Crit Care Med. 36, 2395-2402 (2008).</li><li>Busscher, H. J., 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=Biomaterial-associated+infection:+locating+the+finish+line+in+the+race+for+the+surface.">Biomaterial-associated infection: locating the finish line in the race for the surface.</a> Sci Transl Med. 4, (2012).</li><li>Benard, E. L., 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=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> J Vis Exp. , (2012).</li><li>Carvalho, 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=A+high-throughput+screen+for+tuberculosis+progression.">A high-throughput screen for tuberculosis progression.</a> PLoS One. 6, (2011).</li><li>Veneman, W. J., 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+zebrafish+high+throughput+screening+system+used+for+Staphylococcus+epidermidis+infection+marker+discovery.">A zebrafish high throughput screening system used for Staphylococcus epidermidis infection marker discovery.</a> BMC Genomics. 14, 255 (2013).</li><li>Sar, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+marinum+strains+can+be+divided+into+two+distinct+types+based+on+genetic+diversity+and+virulence.">Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence.</a> Infect Immun. 72, 6306-6312 (2004).</li><li>Stoop, E. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryo+screen+for+mycobacterial+genes+involved+in+the+initiation+of+granuloma+formation+reveals+a+newly+identified+ESX-1+component.">Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component.</a> Disease Model., &amp; Mechanisms. 4, 526-536 (2011).</li><li>Adatto, I., 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+new+system+for+the+rapid+collection+of+large+numbers+of+developmentally+staged+zebrafish+embryos.">A new system for the rapid collection of large numbers of developmentally staged zebrafish embryos.</a> PLoS One. 6, (2011).</li><li>Kimmel, C. B., 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=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Spaink, H. 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=Robotic+injection+of+zebrafish+embryos+for+high-throughput+screening+in+disease+models.">Robotic injection of zebrafish embryos for high-throughput screening in disease models.</a> Methods. 62, 246-254 (2013).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev Biol. 248, 307-318 (2002).</li><li>Mathias, J. 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=Live+imaging+of+chronic+inflammation+caused+by+mutation+of+zebrafish+Hai1.">Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.</a> J Cell Sci. 120, 3372-3383 (2007).</li><li>Henkel, C. V., 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=Comparison+of+the+exomes+of+common+carp+(Cyprinus+carpio)+and+zebrafish+(Danio+rerio).">Comparison of the exomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio).</a> Zebrafish. 9, 59-67 (2012).</li><li>Chang, T. -. Y., 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=Fully+automated+cellular-resolution+vertebrate+screening+platform+with+parallel+animal+processing.">Fully automated cellular-resolution vertebrate screening platform with parallel animal processing.</a> Lab on a Chip. 12, 711 (2012).</li><li>Pardo-Martin, C., 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=High-throughput+in+vivo+vertebrate+screening.">High-throughput in vivo vertebrate screening.</a> Nature Methods. 7, 634-636 (2010).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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 &#181;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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现:斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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. ...

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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

建立和高通量安装程序研究优化

摘要

探索更多视频

此视频中的章节

在细菌粘附的研究剪应力

促进药物发现:斑马鱼在一个自动化的高含量检测炎症

使用

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

高通量设置的建立筛选小分子能调节C-二GMP信令

高通量兼容检测，以评估对巨噬细胞传代药物疗效

分析¹⁸葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

marinum 分枝杆菌感染成人斑马鱼结核的建模

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

一种研究食细胞诱导免疫应答的 mellonella 口服液模型