<p class="jove_content"我们感谢优秀的畜牧业Nadeshda狼，纳丁·埃申和苏珊·德隆。连续由博士研究生奖学金的支持，从亥姆霍兹程序<em&gt; BioInterfaces</em&gt;国际研究生院（BIFIGS）。进一步支持提供了一个工具包-RISC赠款和由玛丽·居里国际重返格兰特在欧洲共同体第七框架计划（PIRG07-GA-2010-267552）到CG。热源支持的BOLD（肝脏和胰腺的发育和疾病的生物学） - 玛丽·居里的初始培训网络（238821）。 UL认证支持Dopaminet第七框架计划（第七框架计划）（223744）。</p&gt;

1。动物处理为下巴实验使用3 DPF幼虫从组纯合的转基因双cldnB :: GFP的/ lysC :: DsRED2和AB型 （野生型）鱼交配，产生的。收集自然产卵胚胎，并提高他们在29°C在E3的介质（5 mM氯化钠，氯化钾0.17毫米，0.33毫米氯化钙 ， 硫酸镁 0.33毫米，0.1％亚甲蓝，平衡pH值7.0），在培养皿中。关键是要保持不超过每盘50-70胚胎密度。 2。幼虫排序根据荧光立体检查所有幼虫荧光记者表达的，自发的炎症和适当的年龄相关的发展。 3。筛选培养基的制备（始终准备好新鲜） 准备不辅以DMSO（最后1％）和MS222（0.05克/升）的亚甲蓝E3中。 4。硫酸铜准备（始终准备好新鲜） 首先掂量出硫酸铜（M R =159.6克/摩尔），并准备了20毫米原液DH 2 O。准备120微米硫酸铜工作在E3/DMSO（1％）/ MS-222解决方案（从20 mm原液）。保护光硫酸铜溶液。 5。 384孔板上下游（格雷纳384微孔板） 预反向移液器每孔加入20μLE3/DMSO（1％）/ MS-222。枪头缸径增加，需要仔细处理，没有造成任何伤人的胚胎，这是由切割2毫米口径的一角。传输介质，在74μL单幼虫，每孔（84μL）未经处理的控制。如果有必要，在卧位东方幼虫在井使用一个灵活的Eppendorf Microloader枪头（Eppendorf公司; 5242 956.003）。 机器人液体进行液体处理步骤处理工作站，以确保所有的幼虫同时治疗。 6。药物治疗吸取了混合药物股板块中的化合物的5倍。每孔加入16μl7.5X药物股板块的和混合的5倍。调整尖内水井的位置，防止幼虫的伤害，并免除在10μL/ s的介质。混合介质井的4倍，以确保药物均匀分布在井。 7。孵化筛选板孵育1小时，在29°C间用铝箔覆盖，避免光线的化合物，以及硫酸铜 。 8。化工伤人除了 ​​阴性对照组，混合4倍，每口井120微米硫酸铜溶液加入10微升，再次孵育1小时29°C。 9。洗涤删除和我交换80μL<html> dium从每个井的两倍（20μL的步骤）删除化合物和硫酸铜 。 10。图像采集自动显微镜倒置（即:奥林巴斯扫描^ R）的图像采集开始90分钟后，初始铜治疗。设置初始Z-级，使左，右后外侧线neuromasts可见。图片每一次每小时渠道明，Cy3和GFP 4焦平面（50微米的距离）使用4倍的目标（，NA = 0.13）。 应要求提供额外的信息，图像和数据处理。 11。影像处理 1。数据整理 RAW图像处理与我们自定义的LabVIEW软件脚本。在图像处理管道的第一个操作是排序显微镜产生的数据通道，以及时间点的信息的文件夹中的原始图像。 ove_step"&gt; 2。扩展的重点随后，该软件创建扩展为每个通道从4焦平面聚焦图像。 3。 RGB-覆盖在最后一步，从3个通道的扩展聚焦图像合并，导致最终的RGB叠加图像。 4。自动神经丘检测一个模式识别工具（LabView的快速IA原型工具）标识内的RGB叠加图像neuromasts和创建经验的兴趣周围neuromasts定义面积。 5。量化红色荧光白细胞（反映为红色像素）内的利息周围受伤neuromasts的的经验定义的区域都取得了主要由 L eukocytes（Paol）中的 p ercent 1 REAØccupied读数。在平均95.27±2.11％（FDA1）和95.12±井（幼虫）（1.56％FDA2）已正确检测并随后受到进一步的数据处理。原始数据输出（Paol为每个检测到的神经丘）被储存在一个txt文件，并作为进一步处理的原始数据的MATLAB脚本的数据输入。 12。数据处理 1。IMAPS 评估个别实验的颜色代码中的原始数据输出Paol图形可视化的成功，可以实现所谓的炎症地图（IMAPS）（ 图2）。明亮的绿色反映了较高的初始炎症指数，黑色表示没有炎症。这种快速概览，允许失败的实验，然后可以排除进一步的数据分析的快速识别。 2。平均控制每个实验板包含320化合物，反映了单个数据点每化合物和时间点以及32个阳性和阴性对照，分别。 32控制重复的平均值和标准偏差的计算。为控制的只有2个标准差之内的数据点都包括在内。 3。正常化正常化是通过电子表格分析。二甲基亚砜的平均值，作为阴性对照，设置为0，和最高的铜控制的的平均Paol是设置为1，因此，阳性和阴性对照之间的最大区别是设置为1。每个化合物的Paol，是线性插值或外推到各自的实验板控制。 4。最后读出:炎症指数后已进行了足够的重复实验（即15），平均归重复实验的原始数据，导致在最后读出 - 炎症索引。铜控制炎症的初始指数开始在100％的时间点零和相关图像采集开始时间（90分钟后，初始铜处理）。 5。单调的指数回归拟合由于解决炎症随着时间的推移，我们对所使用的初始炎症执行单调指数非线性回归拟合<img src="/files/ftp_upload/4203/4203eq1.jpg" alt="式（1）" /> - 0是在t = 0的初始响应的措施，有关斜坡的幅度随着时间的推移。 6。特征空间的2-D图（ 图4） 要生成的功能空间，非线性回归应用于集群分析划分为特征区域的功能空间，从而从不同的免疫调节类有趣的候选人自动识别（反-inflammat的存储器，反决议，亲炎症，有利于决议）。基于参数0和1的2-D图形显示在所有化合物。 13。数据处理和存储我们的数据处理例程创建网页，可视化和代表等药物屏幕，提供了图像的快速概览和数据处理步骤的结果，以及详细视图时，要求用户10。软链接到其他相关的异构生物和化学数据库集成，以及提供了宝贵的资源比较和新颖的研究11。通过这些例程，设置适当的数据标准和元信息这一数据的长期存储。 14。代表结果在试点屏幕上，我们分析了库，包括640 FDA批准启动，进展或决议的麻粒的影响，被称为生物活性化合物ocytic炎症反应。基于我们分为4种类型的免疫调节表型，可能预示不同的行动模式的观测效果:消炎（1），反决议（2），（3）促炎症和亲的决议（4） ，这可以用参数0和1，非单调线性回归拟合（ËAO + ALX - ）产生描述对初始炎症。一个0代表的炎症反应的程度，而1曲线斜率的特点，从而说明炎症决议。一个2-D的功能空间图（0与1）中显示所有的化合物，可以从不同的免疫调节类击中候选人（ 图3）的自动识别。 45出640种减少到50％或更少的初始炎症指数产生显着的抗炎作用。内氏s个分类，我们发现了6个化合物属于药物类的非甾体类消炎药（NSAIDs），证实了我们的方法的有效性，。然而，NSAIDs的排名当中最有力的消炎药。除了从非甾体抗炎药，我们发现了一些额外的药理类药物，如例如血管紧张素Ⅱ受体阻断剂（ARBs），抗生素和质子泵抑制剂。 7个化合物符合为潜在亲分辨率药物的经验定义的阈值标准。 18药物产生了亲消炎作用，在夸张白细胞招聘相比，阳性对照受伤neuromasts。 只有2种化合物，阻止炎症反应的及时解决。 几个候选命中消炎作用（模范候选人从上述药理类药物）从试点屏幕可以变成ð被证实在随后的二次下巴检测（数据未显示）的剂量依赖性。 4个潜在的亲分辨率药物复验未确认的行动施加在主屏幕的指示模式。 2药物在高浓度（20μM）轻微消炎潜力。第三个药物具有非剂量依赖性药物浓度范围从5边际消炎作用 - 20微米。第四药物有没有炎症反应的影响，在测试浓度。 <img alt="图1" src="/files/ftp_upload/4203/4203fig1.jpg" /> 图1。下巴检测工作流程。个人复合基因cldnB :: GFP / lysC :: DsRED2幼虫（3 DPF）手动分布在384孔板。药物被同时添加到每口井使用西风紧凑型液体处理工作站。检测板孵育1小时，在29°C。 硫酸铜治疗</sub&gt; - 解决方案（10微米的最终浓度）1小时，造成伤人和引发急性炎症反应。最后，铜溶液和药物洗掉和自动化图像采集开始初始铜治疗后（90分钟）。 <img alt="图2" src="/files/ftp_upload/4203/4203fig2.jpg" /> 图2。原始图像。概述图片的数字显示原始图像的4焦平面（Z = 0， - 3）在渠道Cy3标记（左）和GFP（右），分别为50微米的距离。箭头（z = 1,2）表示的模范neuromasts，箭镞在neuromasts周围聚集白细胞（z = 1,2）点。 <img alt="图3" src="/files/ftp_upload/4203/4203fig3.jpg" /> 图3。炎症地图（IMAPS）个别实验的成功可以反映炎症反应的颜色代码（原始数据）IMAPS评估:明亮的绿色，体现了高初始infla<html> mmatory响应;黑色表示没有炎症，时间点0（左）和时间点5（右）IMAPS清楚地表明，实验是否应包括在进一步的数据处理。铜对照组（13和24行）显示在深浅不一的绿色，而，二甲基亚砜控制井（1和12行）出现暗绿色或黑色。在时间点5，炎症几乎是解决铜的控制，现在的绿色或黑色的暗色调。 <a href="http://www.jove.com/files/ftp_upload/4203/4203fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4203/4203fig4.jpg" /> 图4。 2维特征空间320 FDA的化合物情节批准库和各自的控制。所有化合物井可以在基于0和1的参数，从一个非线形产生的2-D的特征空间图显示回归拟合（d/4203/4203eq1.jpg"ALT ="公式1"/&gt;对初步炎症）。此功能空间图允许自动识别属于以下4种免疫调节类药物可能指示一个有趣的候选人消炎"的行动模式:（1），抗决议（2），（3）促炎症和亲的决议（4） <a href="http://www.jove.com/files/ftp_upload/4203/4203fig4large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
	<li>Peterson, R. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+use+of+small+molecules+for+probing+biological+processes+in+zebrafish.">Discovery and use of small molecules for probing biological processes in zebrafish.</a> Methods Cell Biol. 76, (2004).</li><li>Zon, L. I., Peterson, R. T. <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+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nat. Rev. Drug. Discov. 4, 35-44 (2005).</li><li>Loynes, C. A., Martin, J. S., Robertson, A., Trushell, D. M., Ingham, P. W., Whyte, M. K., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+advance:+pharmacological+manipulation+of+inflammation+resolution+during+spontaneously+resolving+tissue+neutrophilia+in+the+zebrafish.">Pivotal advance: pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish.</a> J. Leukoc. Biol. 87, 203-212 (2009).</li><li>Martin, J. S., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+in+vivo+zebrafish+models+to+understand+the+biochemical+basis+of+neutrophilic+respiratory+disease.">Using in vivo zebrafish models to understand the biochemical basis of neutrophilic respiratory disease.</a> Biochem. Soc. Trans. 37, 830-837 (2009).</li><li>Renshaw, S. A., Loynes, C. A., Trushell, D. M., Elworthy, S., Ingham, P. W., Whyte, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev. Biol. 7, 42 (2007).</li><li>Redd, M. J., Kelly, G., Dunn, G., Way, M., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+macrophage+chemotaxis+in+vivo:+studies+of+microtubule+function+in+zebrafish+wound+inflammation.">Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation.</a> Cell. Motil. Cytoskeleton. 63, 415-422 (2006).</li><li>D'Alençon, C. A., Peña, O. A., Wittmann, C., Gallardo, V. E., Jones, R. A., Loosli, F., Liebel, U., Grabher, C., Allende, M. L. <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+chemically+induced+inflammation+assay+in+zebrafish.">A high-throughput chemically induced inflammation assay in zebrafish.</a> BMC Biol. 8, 151 (2010).</li><li>Haas, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+signaling+mediates+self-organizing+tissue+migration+in+the+zebrafish+lateral+line.">Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line.</a> Dev Cell. 10, 673-680 (2006).</li><li>Luetjohann, D. S., Shah, A. H., Christen, M. P., Richter, F., Knese, K., Liebel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciencenet'+-+towards+a+global+search+and+share+engine+for+all+scientific+knowledge.">Sciencenet' - towards a global search and share engine for all scientific knowledge.</a> Bioinformatics. 27, 1734-1735 (2011).</li><li>Liebel, U., Kindler, B., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvester':+a+fast+meta+search+engine+of+human+protein+resources.">Harvester': a fast meta search engine of human protein resources.</a> Bioinformatics. 20, 1962-1963 (2004).</li></ol>

没有利益冲突的声明。

<p class="jove_content"&gt;在这里，我们提出了一个完全自动化的高通量筛选策略，使在整个动物中免疫调节化合物的鉴定。从而使急性炎症反应的监测<em&gt;在体内</em&gt;和实时。</p&gt;<p class="jove_content"&gt;我们提供了一个协议的申请，因为白细胞招聘的药物对伤口可以相对容易地在一个功能齐全的先天免疫系统中的研究发现药物或药物再利用的努力作为宝贵的模式生物斑马鱼。我们的方法的独特优势，是一个事实，即化学伤人呈现手册幼虫操纵过时，从而使自动化和筛选，高通量的格式。</p&gt;<p class="jove_content"此外，有害或有毒的药物效果以及强大的脱靶效应可以很容易地评估与建议的设置。然而，FDA批准的库不包含在任何给定的筛选浓度的有毒化合物。尽管如此，我们提出了明显的有毒化合物相同的药理物质测试库中的类中的成员之间的比较可用的数据集（如免疫调节型，药物浓度）。在低浓度有毒物质的家庭，否则对一个有前途的免疫调节型，成员可以进行复检，如果需要的话。</p&gt;<p class="jove_content"我们已经证明自动分类药物的可能性，根据他们施加的免疫调节作用。药物属于类抗炎化合物，可在独立的实验，发挥他们的作用，在剂量依赖性的化合物验证。四个被确定为潜在亲分辨率药物的化合物，可以迄今无法证实等。这也表明这个特定的免疫调节类是很难形容的，因为没有药物仅影响的决议，迄今已在文献中描述。然而，3 4复检毒品的摩尔浓度较高的抗发炎的趋势。优化经验定义的阈值标准应有助于减少误报数量。</p&gt;<p class="jove_content"此外，所提出的技术提供了可能影响白细胞迁徙行为的基因突变的鉴定利用<sup&gt; 8</sup&gt;。</p&gt;<p class="jove_content"&gt;最后，​​该方法可以促进和加快药物发现或药物再利用所产生对药物耐受性，毒性，剂量以及潜在的脱靶效应的信息，随时提供潜在的新的领导结构的努力。因此，作为一个简单的，成本和节省时间，在药物开发的早期阶段战略。</p

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>二甲基亚砜</td><td>卡尔·罗斯有限两合公司</td><td> A994.2 </td><td></td></tr><tr><td>铜（II）无水硫酸钠</td><td>卡尔·罗斯有限两合公司</td><td> PO23.1 </td><td></td></tr><tr><td> FDA批准的药物库</td><td>恩佐生命科学</td><td> BML-2841-0100 </td><td></td></tr><tr><td> 384微孔板的非约束性的，μClear </td><td>格雷纳</td><td> 781906 </td><td>黑色</td></tr><tr><td>奥林巴斯扫描^ R </td><td>奥林巴斯</td><td></td><td></td></tr><tr><td>西风紧凑型液体处理工作站</td><td>卡尺</td><td></td><td></td></tr></tbody>
 </table>

facilitating drug discovery: an automated high-content inflammation assay in zebrafish

斑马鱼幼虫是整个动物特别适合小分子由于其体积小，相对容易操作和观察，以及化合物可以简单地被加入到洗澡水和易被人体吸收，在屏幕1,2 &lt;1％DMSO溶液。由于斑马鱼幼虫和白细胞中表达荧光蛋白的转基因株系的可用性的光学清晰度，斑马鱼体内的一种急性炎症反应的监测提供的独特优势。因此，利用斑马鱼为高含量的小分子，在免疫调节化合物的高通量鉴定屏幕已经提出3-6，表明炎症诱导的情况下，如鱼翅组织本地化的刻痕，蛋黄表面的激光损伤胚胎或尾翼截肢3,5,6。然而，这些方法的主要缺点是幼虫操纵手册的要求，促使伤人，从而防止高通量筛选。化学诱导的炎症（下巴）检测8介绍消除这些障碍。由于伤人是造成化学可以同时处理的胚胎数量几乎是无限的。临时处理的与硫酸铜斑马鱼幼虫的侧线系统和快速粒招募受伤neuromasts的结果毛细胞选择性诱导细胞死亡。随后的炎症反应，可以实时通过复合基因cldnB :: GFP / lysC :: DsRED2 6,9斑马鱼幼虫，表达了绿色荧光蛋白在神经丘细胞，以及红色荧光蛋白标记粒。 为了制定筛查策略，将使高含量和高通量分析，我们介绍了机器人的液体处理和综合自动化MICRoscopy与自定义开发的软件脚本。这个脚本可以自动量化得分％面积的红色荧光白细胞占领周边受伤绿色荧光neuromasts的的经验定义的面积内的炎症反应。此外，自动数据处理，处理，可视化，存储的基础上开发的MATLAB和Python脚本定制。 总之，我们引进一个自动化的HC / HT屏幕测试化合物，允许其对粒细胞炎症反应的启动，进展或决议的影响。该协议提供一个很好的起点，为更深入地分析药物的机制和途径，参与先天免疫反应的编排。在未来，它可以帮助确定不能容忍的有毒或脱靶药物发现的早期阶段的影响，从而降低药物开发的程序上的风险和成本。

Watch this Scientific Journal Video about Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish at JoVE.com

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

在这里，我们描述了一种新型的高含量化学诱导的炎症检测，在免疫调节生物活性鉴定。我们已经成功地结合自动显微镜定制开发的软件，使量化的炎症反应以及进一步的数据处理，分析，挖掘，和存储的自动化脚本。

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 ...

facilitating-drug-discovery-an-automated-high-content-inflammation

Research

JoVE Journal

Immunology and Infection

14.1K 次观看.  Karlsruhe Institute of Technology, Karlsruhe, Germany. 在这里，我们描述了一种新型的高含量化学诱导的炎症检测，在免疫调节生物活性鉴定。我们已经成功地结合自动显微镜定制开发的软件，使量化的炎症反应以及进一步的数据处理，分析，挖掘，和存储的自动化脚本。

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

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

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 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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

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. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

从小鼠在原代肝细胞培养物的炎性反应的诱导

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

一个<em&gt;体外</em&gt; Caseum结合测定，预测药物在渗透结核病变

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...