该NCNPR USDA-ARS科学的协议58-6408-2-0009; CDMRP授予奖＃W81XWH-09-2-0093美国陆军医学研究和装备司令部。

1。维护和继代培养THP1细胞培养<ol><li>维护THP1细胞在RPMI-1640培养基（含10％FBS，pH为7.4）在37℃下在5％CO 2培养箱中。 </li><li>继代培养细胞，每周两次，以防止超过1×10 6细胞/ ml的细胞计数。这是很重要的，保持自己的转型能力。 </li></ol> 2。维护和继代培养的杜氏利什曼原虫前鞭毛体文化<ol><li>保持L.原虫前鞭毛体（S1，苏丹红株）的RPMI-1640培养基（无碳酸氢钠和丙酮酸钠）在26℃与10％FBS的</li><li>继代培养L.原虫前鞭毛体，每周两次，最高细胞浓度范围内20-25×6的前鞭毛体/ ml.Caution至室温，使用前应使所有的媒体和解决方案。 </li></ol> 3。在9播种和分化的THP1细胞6孔微孔板和16室的玻璃文化的幻灯片。 <ol><li>准备一个稀释THP1文化与细胞计数为2.5×10 5细胞/ ml，从4天龄的细胞培养物（细胞计数应不超过10 6细胞/毫升），用10％热灭活FBS的RPMI-1640。制备每个16 - 孔室滑动为每个96孔板中，培养4毫升20毫升培养。 </li><li>佛波醇-12 - 肉豆蔻13-醋酸酯（PMA）（分化的THP1）稀释的细胞培养液（10μl/20毫升培养的股票为50微克/毫升DMSO）（最后的PMA浓度稀释后的细胞培养应该是25纳克/毫升）中。 </li><li>为了比较数字图像分析直接计数检测和寄生虫救援转换的含量，同时检测整洁，平坦的底部，96孔板和16室，玻璃，微观文化的幻灯片。 </li><li>分配200μl的THP1-PMA-处理的细胞的各孔或腔室。 </li><li>孵育96 p鲈和16孔室玻片中，在37℃下，5％CO 2培养箱中培养过夜，允许几乎完全分化的细胞。 </li></ol>注:THP1细胞，通常在悬浮液中生长，分化成粘附的巨噬细胞。 4。感染转THP1细胞杜氏利什曼原虫前鞭毛体<ol><li>差异化THP1细胞培养杜氏利什曼原虫前鞭毛体的感染，大多数的寄生虫的感染的亚循环阶段（长圆筒形的形式，约5-6天旧文化）。 </li><li> 1:10 THP1细胞寄生虫的比例是最佳的数字图像分析直接计数法的前鞭毛虫救援转型含量的感染。 </li><li>准备稀释的文化L.原虫前鞭毛体的寄生虫计数为2.5×10 6寄生虫/毫升（THP1细胞寄生虫的比例为1:10）在含2％FBS的RPMI-1640培养基中的5至6天龄的文化。 </li><li>从步骤3.5（THP1细胞培养过夜后分化）取出的板和分室玻片，除去培养基，并用无血清的RPMI-1640培养基洗细胞培养一次。 </li><li>仔细清洗后PMA处理THP1细胞用无血清，温暖的RPMI-1640（〜37°C）的培养基，用200μl的稀释的培养L.更换无血清培养基原虫前鞭毛体（2.5×10 6个寄生虫/ ml）4.3从步骤。 THP1细胞无寄生虫和寄生虫的控制井没有THP1细胞在每块板和16孔玻片。 </li><li>添加寄生虫THP1细胞培养后，培养板和幻灯片，在37℃，5％CO 2的 24小时以允许寄生虫感染分化THP1细胞。 </li></ol> 5。治疗感染的巨噬细胞与试验药物/化合物<ol><li>测试两性霉素B，喷他脒和米替福新作为标准反leishmanial的药物筛选。准备的药物/试验化合物，在水或DMSO中提到的试剂台的原液。在6浓度测试每种药物的化合物。 </li><li>串行稀释（1:5）的标准药物和试验化合物，在一个新的96 - 孔板或2 ml离心管（用于室玻片）用含2％FBS的RPMI-1640培养基。药物/测试化合物的浓度，在这个板块中的最终浓度的2倍。 </li><li>清洗的文化板和玻片感染，与L.原虫前鞭毛体（来自步骤4.5）至少5次，用无血清的RPMI-1640培养基。 100μl的培养基（含2％FBS的RPMI-1640）添加到各孔/腔室。的多个洗涤是必要的，以确保完全除去的非内化的前鞭毛体。 </li><li>加入100μl连续稀释的标准的反leishmanial药物或测试化合物介质，每孔或室。成立了感染特德THP1细胞没有药物控制，同时在每块板/室幻灯片。 </li><li>孵育板和分室玻片在37℃，5％CO 2的 48小时。 </li></ol> 6。商会幻灯片染色，荧光显微镜成像和图像分析的定量感染和药物治疗效果<ol><li>孵育48小时后，室幻灯片洗3次，用无血清的RPMI-1640培养基。揭去塑料商会从幻灯片中的幻灯片甲醇浸泡30秒，修复细胞。发表在生物罩下的幻灯片气流干燥。 </li><li>准备一个SYBR Green I染色（1:2,000）的股票（10,000 X）与水稀释液（5X）。 </li><li>染色幻灯片与稀释的SYBR Green I的染色溶液在室温下的15分钟，在黑暗条件下与水和洗一次离开幻灯片下的空气流进行干燥。 </li><li>将一个完整的盖玻片在焦虑定义的滑动安装介质的帮助。 </li><li>捕捉数字图像的THP1细胞:没有感染（空），感染（控制），受感染的细胞有不同的标准药物或测试化合物在不同的稀释液（ 图3，5和6））使用的是尼康的Eclipse 90I荧光显微镜伴随着由3.2软件NIS元素A​​R。 </li><li>计数巨噬细胞的细胞核（大）和寄生虫的原子核（小动基）使用ImageJ（ 图7），这是一个公开可用的，基于Java的图像处理程序，在美国国立卫生研究院（ <a href="http://rsb.info.nih.gov/ij/download.html" target="_blank">http://rsb。 info.nih.gov / IJ / download.html</a> ）。，无鞭毛体的数量为每100转化THP1细胞表达数据。 </li></ol> 7。的寄生救援转型含量:对照裂解L.原虫无鞭毛体感染的巨噬细胞<ol><li>洗净的96孔微孔板中，来自步骤5.5三次用无血清的RPMI-1640培养基中。 </li><li>在不同的清洁剂测试在不同的浓度，0.05％SDS处理30秒是最佳的控制裂解（最大细胞裂解救出寄生虫的生存损失降到最小）。 </li><li>最后一次洗涤后，从每个孔中取出的无血清培养基，向每孔中加入20μl的RPMI-1640（0.05％SDS）。摇动板30秒，向每孔加入180微升的完全RPMI-1640培养液（含10％FBS）。 </li><li> 48小时获救无鞭毛体前鞭毛体转化，孵育板在26°C。 </li></ol> 8。转化前鞭毛虫（定量分析的AlamarBlue检测） <ol><li>经过48小时的潜伏期在26°C，所有获救的活无鞭毛体转化为前鞭毛体（ 图1D）。向每个孔中加入10微升的AlamarBlue 96孔p尖吻鲈。 </li><li>将培养板在26°C过夜。 </li><li>培养过夜后，读的（BMG实验室技术）在544 nm激发下，590nm的发射一个Fluostar银河荧光计上的标准荧光板。 </li><li>准备的剂量响应曲线（％的增长与浓度的药物或试验化合物）与ExcelFit（ 图7-9）和计算IC 50 /从这些曲线中的IC 90值（ 表1）。 </li></ol> 9。标准化的THP1细胞中的寄生虫比<ol><li>规范THP1细胞寄生虫的比率，以确定检测的灵敏度。注:寄生虫数字低/高/感染可能会影响灵敏度和选择性的筛选。 </li><li>规范THP1细胞寄生虫的比率由上述协议后THP1细胞播种和利什曼原虫前鞭毛虫感染（第3和4）除了使用different的THP1细胞比例为1:1.25，1:2.5，1:5和1:10的寄生虫。 </li><li>设置实验在16室的滑动件（用于图像分析）和96孔板中（寄生虫救援测定）格式，用来比较的寄生虫救援和图像分析检测。 </li></ol> 10。不同的清洁剂来控制细胞裂解标准化<ol><li>这个实验的主要目标是优化THP1细胞，以达到最大/完整的THP1细胞裂解裂解控制没有显着影响的可行性获救的无鞭毛体寄生虫的协议。 </li><li>测试在不同浓度和不同的持续时间的处理（ 图2）不同的洗涤剂，如吐温20，吐温80，曲拉通X-100，NP-40和SDS。 </li><li> THP1细胞寄生虫的比率是1:10和其他条件类似上述部分1-8。 </li><li>不同处理时间进一步测试0.05％SDS处理s的进一步优化的控制细胞裂解。 </li></ol>

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没有利益冲突的声明。

有几种方法可用于基于巨噬细胞的无鞭毛体模型的的反leishmanial药物筛选。检测可以做到与收集从宿主动物即腹膜渗出细胞（PEC），外周血单核细胞（PBMC）的巨噬细胞6或骨髓来源的巨噬细胞（BMM）或单核细胞的细胞系，例如鼠标（J774和RAW264.7 ）7和人类（THP1，U937，HL-60）8单核细胞。使用的分析，将宿主细胞，必须保证药物活性的影响因子，被认为是寄生虫与宿主细胞的数量。收集来自各种来源（如小鼠和大鼠）的巨噬细胞分化的初级非分裂的性质，但这些细胞制剂可能不具有均匀的细胞群。单核细胞衍生的细胞系在性质上是同质的，是一个更好的模型的巨噬细胞无鞭毛体为基础的筛选。在不同的单核细胞系，不同rentiated THP1细胞（人急性单核细胞白血病细胞系），可以形成非分割的单层和一次分离巨噬细胞提供了一个有吸引力的替代。 无鞭毛体的巨噬细胞为基础的筛查，可以做到在几个方面。古典微观评价的基础上直接的细胞和寄生虫计数9是劳动密集型的。自动化的情况下限制该测定中的效用。细胞计数是耗时，并可能给出不准确的IC 50值的测定，因为测定的寄生虫生存能力通过染色程序是困难的。许多荧光染料的抗体和单克隆抗体可用于流式细胞术分析10，11，但这些测定也有限的，由于较低的灵敏度和限制的药物处理的时间间隔只有一天。有几个报告基因检测可用于定量细胞内的无鞭毛体的生长12,13,14。一个自动贩卖机编筛选可能可以使用的报告基因，但这些测定也有某些缺点。首先，大多数这些实验需要保持游离的报告基因的表达，这可能不是理想的药物筛选实验药物的选择。报告基因的引入方式，其中也可能影响生理特性的寄生虫，并有一定影响的筛选。如果报道基因是附加体的质粒的一部分，相对输出的记者可能依赖于转染的质粒的拷贝数（从细胞到细胞而变化），而不是上的活性的药物14。某些记者转化寄生虫寄生虫不需要选择性的压力，以保持报道基因，然而，有可能是生物的后果可以通过破坏的基因组的体系结构或只是由外国记者蛋白15的存在下。在一些报告基因为基础的分析，有问题的敏感性和背景活动16。最重要的是，许多报道基因表达分析，特别是1用GFP记者gene15，可能无法区分活的和死的细胞内的无鞭毛体。检测荧光素酶报告基因的基础上可分辨活的和死的细胞内的无鞭毛体，但这些测定的基板和细胞溶解缓冲液是昂贵的大规模筛选17。为了克服这些缺点和局限性的巨噬细胞无鞭毛体为基础的筛选试验，我们已经开发和优化这种寄生虫救援和转化实验。此测定是基于THP1细胞，它具有良好的均匀性和非分裂的性质，作为宿主细胞。 这里描述的是寄生虫的救援转型含量检测的分析基于数字图像分析直接计数细胞内的无鞭毛体的相媲美。荧光和DIC显微镜，数码成像ImageJ的差分计数的巨噬细胞的细胞核和寄生虫的核戊分析，进一步完善了显微计数分析。荧光灯下的过滤器和微分干涉相差（DIC）的过滤器捕获的图像的改进的数字图像的质量，以便更准确地计数的细胞内寄生虫。荧光和DIC图像可以合并获得的数字图像与清晰的巨噬细胞的轮廓和荧光细胞内的细胞核。巨噬细胞的细胞核和寄生虫核差异与ImageJ的认可。因此，这两个数字图像分析直接计数检测和寄生虫救援转换的含量有可能大规模筛选自动化和应用程序。寄生救援-转化-含量中的关键步骤是:（一）暴露在利什曼原虫前鞭毛体的的THP1细胞培养后，反复洗涤，以确保几乎完全除去的非实习生alized前鞭毛体和（b）控制的感染THP1细胞与SDS裂解。这两个步骤也可以被控制与自动化和不应妥协测定的吞吐量。利什曼原虫的感染THP1细胞暴露试验药物/化合物的第二个步骤洗涤后，去除剩余的非内在的寄生虫，如果有的话。寄生虫的救援转型含量提供显着的优势，在现有的微观，报告基因和图像分析检测。该法是简单，可靠，重现性好，可以自动进行大规模的筛选，因此应为新的反leishmanial的药物发现的大分子化合物库中筛选有重要的应用。另外，也可以被应用于测定感染性临床评价，以及，实验室株利什曼原虫在体外 。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>佛波醇-12 - 肉豆蔻13 - 醋酸酯（PMA） </td><td> Sigma-Aldrich公司（美国） </td><td> P1585 </td><td>所需的THP1细胞的分化。 </td></tr><tr><td>的RPMI-1640中等</td><td> Invitrogen公司</td><td> 23400021 </td><td></td></tr><tr><td>实验室-Tek腔室滑动系统（16室） </td><td>赛默飞世尔科技Nunc公司</td><td> 178599 </td><td></td></tr><tr><td>清除，平底，96孔板</td><td> BD猎鹰</td><td> 353075 </td><td>板来执行96 - 孔板检测</td></tr><tr><td>两性霉素B </td><td> Sigma-Aldrich公司（美国） </td><td> A4888 </td><td>标准的反leishmanial药物溶解在DMSO（2毫克/毫升） </td></tr><tr><td>喷他脒异硫代萨&lt; </td><td> Sigma-Aldrich公司（美国） </td><td> P 0547 </td><td>标准的反leishmanial药物溶解在DMSO（2毫克/毫升） </td></tr><tr><td>米替福新</td><td>美国EMD Biosciences公司</td><td> 475841 </td><td>标准的反leishmanial药物溶解在无血清的RPMI-1640培养基（准备新鲜的）（2毫克/毫升） </td></tr><tr><td>葡萄糖酸锑钠</td><td>美国EMD Biosciences公司</td><td> 567565 </td><td>与4％FBS的RPMI-1640标准的反leishmanial药物准备。 （1毫克/毫升） </td></tr><tr><td>十二烷基硫酸钠</td><td> Sigma-Aldrich公司（美国） </td><td> ł5750 </td><td></td></tr><tr><td>荧光显微镜</td><td> NIKON </td><td> ECLIPSE 90I </td><td></td></tr><tr><td>荧光酶标仪</td><td> BMG </td><td>极星驰</td><td></td></tr><tr><td>安装中</td><td> Sigma-Aldrich公司（美国） </td><td>中号1289 </td><td></td></tr><tr><td>一lamarBlue </td><td> ABD Serotec公司</td><td> BUF012乙</td><td></td></tr><tr><td> SYBR Green I的</td><td> Sigma-Aldrich公司（美国） </td><td>小号9430 </td><td></td></tr><tr><td>碳酸氢钠</td><td>费舍尔科学。 </td><td> 523500 </td><td></td></tr><tr><td>丙酮酸钠</td><td> Sigma-Aldrich公司（美国） </td><td> P2256 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Sigma-Aldrich公司（美国） </td><td> M6250 </td><td></td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11050H </td><td></td></tr><tr><td>吐温20 </td><td> Sigma-Aldrich公司（美国） </td><td> P9416 </td><td></td></tr><tr><td>吐温80 </td><td> Sigma-Aldrich公司（美国） </td><td> P6474 </td><td></td></tr><tr><td>的Triton X-100的</td><td> Sigma-Aldrich公司（美国） </td><td> T8787 </td><td></td></tr><tr><td> NP-40 </td><td> Calbiochem公司</tD&gt; <td> 492016 </td><td></td></tr></tbody></table>

a parasite rescue and transformation assay for antileishmanial screening against intracellular leishmania donovani amastigotes in thp1 human acute monocytic leukemia cell line

利什曼病是世界上最被忽视的疾病之一，主要是影响到最贫穷的人，主要是在发展中国家。有超过350万人被认为是感染利什曼病的风险，约2万新发病例，每年发生1。 杜氏利什曼原虫的病原体为内脏利什曼病（VL），最致命的疾病。 2可用于治疗利什曼病的药物的选择是有限的，目前的治疗方法治疗效果有限，许多是有毒的治疗剂量。另外，大多数的第一线治疗药物已经失去了其效用由于增加多重耐药性3。目前管道的反leishmanial药物也严重枯竭。持续的努力来丰富一个新的反leishmanial的药物发现管道，努力依赖于是否有合适的体外筛选模型。 在体外的前鞭毛体和无菌无鞭毛体检测主要用于的反leishmanial药物筛选，但由于显着的细胞，生理，生化和分子细胞内的无鞭毛体比较差异，可能不适合。检测与巨噬细胞的无鞭毛体模型被认为是最接近的利什曼病的病理生理条件下，因此是最合适的体外筛选。有区别的，非分割的人类急性单核细胞白血病细胞（THP1），（使一个有吸引力）替代隔离的巨噬细胞，并可以用于不同的化合物对细胞内的无鞭毛体测定的反leishmanial活性。 在这里，我们提出了一个的寄生虫救援和转化实验感染杜氏利什曼原虫的体外筛选纯化合物和天然产品的差异化THP1细胞的UCTS提取物和确定的疗效，对细胞内的利什曼原虫无鞭毛体。该试验涉及以下步骤:（1）THP1细胞的非分割的巨噬细胞的分化的影响，（2）感染的巨噬细胞与L.亚循环前鞭毛原虫 ，（3）治疗感染的细胞与试验药物，（4）控制感染的巨噬细胞溶解，（5）无鞭毛体（6）改造现场无鞭毛体前鞭毛体的释放/救援。该法进行了优化，使用清洁剂处理，控制利什曼原虫感染的THP1细胞的裂解，实现几乎完全的拯救他们的能力，转化为前鞭毛体的活细胞内的无鞭毛体的影响最小。不同的巨噬细胞:前鞭毛体的比例进行了测试，达到最大的感染。量化的感染进行改造现场，救出了利什曼原虫无鞭毛体前鞭毛体和评估其增长的阿拉姆arBlue荧光检测在96孔微孔板中。该法是与目前所使用的显微镜​​下，转基因报告基因和数字图像分析测定。此法是健全和措施，只有在活细胞内的无鞭毛体相比，报告基因和图像分析检测，可能无法区分活的和死的无鞭毛体。另外，检测已被验证的电流面板反leishmanial药物，并已成功地应用于大规模筛选的纯化合物和天然产物的馏分（Tekwani 等人，未出版）图书馆。

两个数字图像分析直接计数检测和的寄生虫救援转型含量进行了定量分析为24，48，72和96小时后药物治疗在直接计数法，感染L.原虫感染的巨噬细胞（THP1细胞）是由下面的等式计算: <img alt="式（1）" fo:content-width="3.75in" fo:src="/files/ftp_upload/4054/4054eq1highres.jpg" src="/files/ftp_upload/4054/4054eq1.jpg" />无鞭毛体（通过无鞭毛体细胞核计数确定）/ 100转化THP1细胞（确定在THP1细胞核计数计数）（ 图7）是更准确的测量，分析不同的标准或试验化合物的影响比受感染的THP1细胞的百分比，以前的一些论文中所报告的，因为这个数字是直接关系到整体效果的化合物，无论是通过减少计算寄生在巨噬细胞或巨噬细胞cells.Infection总去除寄生虫感染的不同稀释度的不同的时间间隔（ 图10和表1）与不同的标准药物治疗在THP1细胞从数字图像。读出的数字图像分析直接计数-含量无鞭毛体infection/100转化THP1细胞，而读出的寄生救援-转化-含量的时间是相对荧光单位（RFU），这是获救从感染的巨噬细胞的活利什曼原虫的无鞭毛体的数目成正比，并变换成前鞭毛体。经常使用的AlamarBlue检测利什曼原虫前鞭毛体的反leishmanial药物筛选。 该法最初是规范和优化控制利什曼原虫感染的THP1细胞裂解。其目的是要优化的条件用于洗涤剂特雷atment，收率​​THP1细胞几乎完全溶解的影响最小获救的无鞭毛体的可行性。 图1示出了完整的检测协议的微观图。完好无损THP1细胞感染利什曼原虫的无鞭毛体，可以看到在图1A，图1B示出的洗涤剂处理后THP1细胞裂解。 图1C示出获救利什曼原虫无鞭毛体，其中部分已被转化为前鞭毛体和图1D示出了几乎完全转化的无鞭毛体进入前鞭毛体，并随后将其增殖。这些转化的前鞭毛体的生长可以定量监测与添加的AlamarBlue和测量的荧光酶标仪。治疗与NP-40（ 图2A）和Triton X-100的（ 图2B）裂解感染THP1细胞，但是，它也影响的生存能力和获救的无鞭毛体的改造。含吐温20（ 图2C）和吐温80（ 图2D）的处理没有引起导致到一个不完整的无鞭毛体救援THP1细胞，如转化的前鞭毛体的低的数字所表示的最佳的溶解。治疗与0.05％SDS，30秒（ 图2E），得到几乎完全裂解Leishmania感染THP1细胞，并没有影响获救无鞭毛体的可行性和改造。进一步的优化表明，处理过的细胞用0.05％SDS的20-30秒得到可行的利什曼原虫的无鞭毛体（ 图2F）的最高救援。在随后的实验中，用0.05％SDS的治疗，持续30秒。 SDS处理的程序是相同的单个或多个板。在多板，SDS处理实施柱柱多道移液器。除去无血清培养基中，从板的一列的所有8个孔的20＆亩; l的0.05％SDS的溶液中加入的同一列中的8个孔中，并与10％FBS的RPMI-1640稀释，在30秒后。测定初始标准化的期间，将板非内化的前鞭毛体在显微镜下检查。至少五个洗涤是必要的以除去寄生虫感染的巨噬细胞与标准化合物和洗涤三次SDS处理的步骤7的前有必要的治疗在步骤5之前。因此，将细胞洗涤8次，并没有可见的非内化的前鞭毛体保持在控制感染THP1细胞裂解之前。 数字图像分析和直接计数的数字图像捕获Leishmania感染THP1细胞有关Nikon Eclipse的90I荧光显微镜下观察与特征动质体DNA的荧光过滤器这两种巨噬细胞的细胞核和细胞内的利什曼原虫细胞核染色后用SYBR Green I.（ 图3）。另外，摄影图像的感染THP1细胞也被下拍摄DIC。当两个图像的合并，THP1细胞与细胞内的无鞭毛体的轮廓更清楚地看出（ 图4）。的的ImageJ软件，用来分析这些图像。 ImageJ是一个公共领域的，基于Java的图像处理程序在国家机构的健康（ <a href="http://rsb.info.nih.gov/ij/download.html" target="_blank">http://rsb.info.nih.gov/ij/download.html</a> ）开发的。 ImageJ的设计已通过Java插件和录制的宏提供了可扩展的开放式架构。自定义的采集，分析和处理插件，可开发利用ImageJ的内置的编辑器和Java编译器。对于差分计数的THP1细胞的细胞核和寄生虫核的ImageJ的，图像被打开了ImageJ的。细胞的计数器被发现在分析选项的软件插件。图像进行初始化和细胞计数类型被选定为THP1Ç埃尔细胞核和细胞计数器类型2被选择的寄生虫核（ 图3）。做了至少200 THP1细胞的细胞核和细胞内的无鞭毛体，THP1细胞的细胞核中存在的这些差异点票。寄生虫救援法和图像分析方法比较了具有不同的巨噬细胞的前鞭毛体的比例（ 图4）用于评估THP1细胞的感染性， 图5表示的差分THP1细胞中的感染，在不同的巨噬细胞:前鞭毛虫比率。两种方法都显示，可比的结果和巨噬细胞:前鞭毛虫的比例为1:10，取得了最佳的和可重复的感染力。 一旦条件parasite-rescue/transformation法和数字图像分析进行了优化，该实用程序的这些测定评价为反leishmanial药物筛选。 利什曼原虫感染的THP1细胞用不同浓度的标准一NTI-leishmanial药物，即两性霉素B，喷他脒和不同的时间间隔为24至96小时，米替福新。实验寄生虫救出/转化试验中，一式三份进行，并直接细胞计数法进行一式两份的实验， 图6示出了控制感染，控制感染未处理和利什曼原虫感染，治疗THP1细胞的显微镜图像。的剂量响应曲线，从寄生虫救援和转化实验（对比转化寄生虫药物的浓度）和图像分析测定（amastigotes/100 THP1细胞的数目）（图7-9）中制备。的药物的IC 50分别计算通过ExcelFit和列于表1中。数字图像分析直接计数检测和寄生虫救援转型含量的显示，可比的结果。数字图像分析直接计数法得到优化的较少，在早期时间点的24小时和48小时药物吨reatments，而寄生救援转型检测结果显示结果更符合报告的值在所有的时间点在24-96小时后，药物治疗。结果与数字图像分析直接计数检测和寄生虫救援转型含量的差异可能是由于存在非可行的无鞭毛体在早期的药物治疗期间的数字图像分析直接计数 - 检测。 <img alt="图1" src="/files/ftp_upload/4054/4054fig1.jpg" /> 图1。显微镜观察杜氏利什曼原虫无鞭毛体救援和改造前鞭毛体。 A -贴壁THP1细胞感染了利什曼原虫无鞭毛体; B -贴壁，受感染的THP1细胞，在控制裂解Ç -救出感染THP1巨噬细胞的电池D的无鞭毛体转化杜氏利什曼原虫前鞭毛体-细胞生长和增殖的转录sformed 杜氏利什曼原虫前鞭毛体。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/4054/4054fig2highres.jpg" src="/files/ftp_upload/4054/4054fig2.jpg" /> 图2。优化的控制感染的THP1细胞裂解，以实现最大的救援现场杜氏利什曼原虫无鞭毛体和转型前鞭毛体。THP1细胞和救援的无鞭毛体，从不同的清洁剂利什曼原虫感染的 THP1细胞裂解分析。两个的洗涤剂的浓度（0.05％和0.1％）和两个时间周期（30秒和60秒），用于治疗进行了测试。 RFU相对荧光单位。每个酒吧的平均重复观测。[A] NP-40治疗引起的THP1细胞裂解，也影响了获救的无鞭毛体寄生虫的可行性。[B]的Triton X-100疗程NT引起的THP1细胞裂解，也影响了获救的无鞭毛体寄生虫的可行性[C]吐温80的THP1细胞引起的部分裂解抢救无鞭毛体。[D]吐温80的THP1细胞引起的部分裂解抢救无鞭毛体。[E [SDS处理几乎完全裂解引起的THP1细胞获救的无鞭毛体的可行性，并没有影响在0.05％/ 30秒[F] 20-30秒，0.05％SDS的治疗造成了几乎完整的THP1细胞裂解，并救出可行的寄生虫无鞭毛体转化为前鞭毛体。 <a href="http://www.jove.com/files/ftp_upload/4054/4054fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4054/4054fig3.jpg" /> 图3。荧光数字图像的分化的THP1细胞在体外感染Leishmani原虫无鞭毛体。也可以看出的特征kDNA与每个寄生虫核。巨噬细胞的核（MN）（1）和寄生虫核（PN）（2）差异标记和差分计数ImageJ的分析软件进行定量评价的感染。定量的amastigotes/100 THP1细胞数。 <img alt="图4" src="/files/ftp_upload/4054/4054fig4.jpg" /> 图4。比较数字图像分析直接计数含量（下图）和寄生虫救援转型含量（上图）。的巨噬细胞:前鞭毛虫之比为1:10，得到最佳的感染。这两个人都表现出类似的结果。救援的寄生虫检测结果显示一些背景值。每个栏显示重复值的意思。 <img alt="图5" srC =的"/ files/ftp_upload/4054/4054fig5.jpg"/&gt; 图5。THP1细胞感染利什曼原虫前鞭毛体由不同THP1:前鞭毛虫比。作为amastigotes/100 THP1细胞数的定量结果在图4中给出。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/4054/4054fig6highres.jpg" src="/files/ftp_upload/4054/4054fig6.jpg" /> 图6。数字图像（从萤光灯+ DIC）的THP1细胞感染利什曼原虫无鞭毛体与标准的反leishmanial药物治疗后不同的时间段。 结果进行了量化的作为中的amastigotes/100 THP1细胞数，并用于计算％的增长，与未处理的对照组相比，确定IC 50值。 <img alt="图7" fo内容宽度="4.75in"佛:SRC ="/ files/ftp_upload/4054/4054fig7highres.jpg"SRC ="/ files/ftp_upload/4054/4054fig7.jpg"/&gt; 图7。比较数字图像分析直接计数检测和寄生虫救援转型为的反leishmanial药物筛选（Amophotericin B）的测定。治疗感染的巨噬细胞与不同浓度的标准抗leishmanial药物的不同时期。 IC 50值（微克/毫升）计算出的剂量反应曲线Excelfit。 <a href="http://www.jove.com/files/ftp_upload/4054/4054fig7large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图8" fo:content-width="4.75in" fo:src="/files/ftp_upload/4054/4054fig8highres.jpg" src="/files/ftp_upload/4054/4054fig8.jpg" /> 数字图像分析直接计数检测和寄生虫救援转型含量的比较图8。的药物筛选NTI-leishmanial（喷他脒）。治疗感染的巨噬细胞与不同浓度的标准抗leishmanial药物的不同时期。 IC 50值（微克/毫升）计算出的剂量反应曲线Excelfit。 <a href="http://www.jove.com/files/ftp_upload/4054/4054fig8large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图9" fo:content-width="4.5in" fo:src="/files/ftp_upload/4054/4054fig9highres.jpg" src="/files/ftp_upload/4054/4054fig9.jpg" /> 图9比较数字图像分析直接计数法和反leishmanial药物筛选（Mitefosine）的寄生虫救援转型含量。被感染的巨噬细胞分别用不同浓度的标准的反leishmanial药物不同的时间段。从Excelfit的剂量响应曲线计算IC 50值（微克/毫升）。<a href="http://www.jove.com/files/ftp_upload/4054/4054fig9large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr><td rowspan="2">测试药物</td><td colspan="2"> 24小时一 </td><td colspan="2"> 48小时一 </td><td colspan="2"> 72小时一 </td><td colspan="2"> 96小时一 </td></tr><tr><td> IACA b </td><td> PRTAÇ </td><td> IACA b </td><td> PRTAÇ </td><td> IACA b </td><td> PRTAÇ </td><td> IACA b </td><td> PRTAÇ </td></tr><tr><td>两性霉素B </td><td> 0.24±0.03 </td><td> 0.17±0.01 * </td><td> 0.12±0.04 </td><td> 0.20±0.07 </td><td> 0.06±0.01 </td><td> 0.06±0.01 </td><td> 0.11±0.03 </td><td> 0.10±0.03 </td></tr><tr><td>喷他脒</td><td> &gt; 10 </td><td> 2.55±1.16 * </td><td> 2.88和PLusmn 0.58 </td><td> 1.43±0.91 </td><td> 1.24±0.35 </td><td> 1.52±0.16 </td><td> 0.71±0.63 </td><td> 0.98±0.33 </td></tr><tr><td>米替福新</td><td> 0.38±0.02 </td><td> 0.19±0.08 * </td><td> 0.24±0.06 </td><td> 0.30±0.08 </td><td> 0.36±0.02 </td><td> 0.16±0.06 </td><td> 0.21±0.15 </td><td> 0.17±0.10 </td></tr></tbody></table> 表1的数字图像分析直接计数检测和寄生虫救援转型含量为的反leishmanial药物筛选。治疗感染的巨噬细胞与不同浓度的标准抗leishmanial药物的不同时期。从剂量反应曲线计算的IC 50值（微克/毫升） 一个小时后的药物治疗，B IACA =图像分析和直接计数者Excelfit（ 图7-9）。含量，C PRTA寄生虫救援和转化实验。所给出的值是为微克/毫升的IC 50值 （引起50％抑制寄生虫的生长的药物的浓度）是至少三次实验的平均值±SD。 *显着性差异（P &lt;0.05）相比，IC 50值IACA。

Watch this Scientific Journal Video about A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic L

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

THP1细胞感染寄生虫救援和转化试验<em在体外</em&gt;与<em&gt;杜氏利什曼原虫</em进行了优化为反leishmanial药物的筛选。该法涉及的THP1细胞，前鞭毛体感染，治疗与试验药物，控制受感染的巨噬细胞的溶解，抢救无鞭毛体，前鞭毛体的改造和监控前鞭毛虫的生长和增殖与荧光检测的分化。

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

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Research

JoVE Journal

Immunology and Infection

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

22.1K 次观看.  University of Mississippi. THP1细胞感染寄生虫救援和转化试验

视频: A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

在仓鼠的背部皮肤:皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

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 ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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 ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

利用人类诱导的多能干细胞衍生的肠道有机体研究和改性上皮细胞对沙门氏菌和其他病原体的保护

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial&#160;propagation.

The recent epidemic of Zika virus highlights the importance of establishing reverse genetic approaches to develop vaccines and/or therapeutic strategies. Here, we describe the protocol to rescue an infectious recombinant Zika virus from a full-length cDNA clone assembled in a bacterial artificial chromosome under the control of the human cytomegalovirus immediate-early promoter.

从细菌人工染色体cDNA克隆中拯救重组寨卡病毒

The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines ...