This work was supported by grants 51RT 0_126008 and 51RTP0_151029 for the Research and Technology Development (RTD) project InfectX and TargetInfectX, respectively, in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology. We acknowledge grant 310030B_149886 from the Swiss National Science Foundation (SNSF). Work of S.H.L and A.C. was supported by the International PhD Program "Fellowships for Excellence" of the Biozentrum. Simone Muntwiler is acknowledged for technical assistance. We would like to thank Dirk Bumann for providing pNF106 and Jean Celli for pJC43 and pJC44.

注意:用活流产布鲁氏菌菌株的所有工作必须考虑到所有必要的法规和安全防范生物安全3级（BSL3）实验室内进行。 1.筛选板的制备和细菌细胞的培养<ol><li> 流产布鲁氏菌发酵剂的制备<ol><li>条纹流产布鲁氏菌在含有50μg/ ml卡那霉素（TSA /公里）一个胰酶大豆琼脂（TSA）平板2308（B.流产 ）-80℃的牛奶库存。在37℃下孵育板3-4天。采用应变流产布鲁氏菌 2308 pJC43（apHT :: GFP）13端点检测和应变流产布鲁氏菌 pAC042.08（apht ::红色荧光蛋白，TETO ::四面体-GFP）的入口检测。 注意: 流产布鲁氏菌的光滑脂多糖（LPS）是一个重要的毒力决定，但粗糙突变体可以在相对 ​​高的频率14发生。 W¯¯Ë因此建议用这个实验启动前测试应变文化的地位。 </li><li>四个TSA /公里板Restreak细菌覆盖全板，并在37°C孵育2-3天。 </li><li>使用从TSA /公里板在10毫升10％灭菌脱脂牛奶一次性塑料环，转让和悬浮细菌。确保细菌以及悬浮以确保在所有发酵剂培养物一致的细菌浓度。 </li><li>等分试样将250μl细菌悬浮液中于2ml螺旋盖试管并冷冻在-80℃。 </li><li>解冻起始培养物的等分试样，并转移25微升，50微升，100微升细菌起子培养分开加入250ml螺旋盖瓶用50ml胰蛋白酶大豆肉汤（TSB）中含有50μg/ ml卡那霉素（TSB /公里） 。密封用封口膜的瓶子。 </li><li>过夜孵育以100rpm和37℃的轨道振荡器的瓶子。 </li><li>测量在600 n中的光密度米（OD 600），以确定过夜培养后达到的OD 600 = 0.8-1.1所需起始培养的体积。 </li></ol></li><li> HeLa细胞的制备<ol><li>生长的HeLa细胞在Dulbecco改进的在37℃的潮湿培养箱中在补充有10％胎牛血清（FCS）（DMEM / 10％FCS）中，用5％CO 2生长细胞至80％汇合的Eagle培养基（DMEM）中。 </li></ol></li><li>准备与siRNA的隔离板。 <ol><li>稀释在无RNase水的siRNA到0.32μM的终浓度。转印5微升/孔黑色透明孔平底384-孔板。 <ol><li>使用，列1 2，23和24的标准控件。对于阴性对照，使用非靶向siRNA（加扰）和模拟井无siRNA（即仅转染试剂）。用于转染的控制，可使用能诱导细胞死亡的siRNA（KIF11），以及涉及在布鲁氏菌感染（ 例如 ，ARPC3已知宿主因素的阳性对照，参与肌动蛋白聚合的Arp2 / 3复合）的一个组成部分。转移市售的siRNA库或自定义的siRNA到其余的孔中。 </li><li>密封板可剥离的铝箔​​。在-20℃储存板。 注意:板可以储存在-20℃至少6个月，至根据制造商的建议年。 </li></ol></li></ol></li></ol> 2.反向siRNA转染<ol><li>在300 XG解冻离心黑色384孔板，在室温下20分钟。 注意:这将降低所有可能粘附到盖子或孔的一侧的液体。 </li><li>在DMEM 200无FCS在室温下:通过稀释转染试剂1准备转染培养基。使用比现有20分钟不再准备转染培养基。仔细混合使用前解决方案。 </li><li>添加25微升转染培养基到每个使用试剂分配器井并且通过移动该板来回混合溶液。 </li><li>孵育1小时的板在室温下，以允许的siRNA /转染试剂复合物的形成。 </li><li>在此期间，通过用2.5ml的0.05％胰蛋白酶-EDTA的PBS（胰蛋白酶）洗涤一75cm 2的烧瓶中的子融合细胞一旦制备HeLa细胞。 </li><li>添加1.5毫升新鲜胰蛋白酶，将烧瓶转移到37℃2-3分钟，直到细胞围捕。 </li><li>悬浮细胞在10ml预热的DMEM / 16％FCS中。 </li><li>计数细胞使用自动细胞计数器和制备的在DMEM / 16％FCS中10,000个细胞/ ml的细胞悬浮液。 </li><li>加入50μl细胞悬液，每孔用导致500个细胞/孔的试剂分配器。 </li><li>移动板来回实现甚至细胞的分布。留在室温下将板5-10分钟，以允许细胞定居下来。 </li><li>用封口膜密封该板孵育它72小时上的预热铝板在5％的CO 2中的37℃湿润培养箱中。 注意:预热铝板允许在整个384孔板相等的温度分布。 </li></ol> 3.感染与固定<ol><li>感染前一天，接种在B的1.1.7确定的量在250毫升50毫升TSB /公里介质流产起子培养螺丝帽瓶中。密封该瓶用Parafilm和在37℃和100rpm的轨道摇床过夜生长的细菌OD 600 = 0.8-1.1。 </li><li>测量细菌培养的外径600和通过稀释细菌培养在DMEM / 10％FCS中，以达到感染复数（MOI）的所需多样性制备感染培养基。 注意:要获得在HeLa细胞5-10％的感染率，10000的MOI使用。一个MOI滴定曲线应为每种细胞类型，以获得最佳的感染率来执行。 </li><li>交换384孔板的转染培养基用50微升感染培养基的使用自动板洗涤器。 </li><li>离心384孔板在400 xg离心4℃20分钟。 注意:这有助于同步感染过程。 </li><li>用封口膜密封该板，并用5％的CO 2孵育其放在预热的铝板在37℃的潮湿培养箱中培养4小时。 </li><li>用含有100微克/毫升了Gm使用自动洗板灭活胞菌的DMEM / 10％FCS中洗涤细胞（使用200μl/孔溢出清洗）。 注:在溢出清洗，自动洗板机同时免除和抽吸介质。 </li><li>有关条目测定法中，洗涤细胞用含有100微克/毫升Gm与100纳克/毫升的aTc使用自动洗板机的DMEM / 10％FCS中的第二时间（使用200μl/孔溢出清洗）。 </li><li>用封口膜密封该板，并在37℃湿润培养箱中，用5％CO 2的另一个它返回到预热的铝板40小时或4小时，分别在端点检测和入口测定。 </li><li>为固定，使用自动洗板（使用200μl/孔溢出洗涤）洗涤各孔用PBS。 </li><li>交换的PBS用自动洗板机中的0.2M HEPES 50微升3.7％多聚甲醛在pH7.4（PFA）。 注:警告:PFA是吸入有毒的，与皮肤接触及吞食。 </li><li>孵育在室温下20分钟。 </li><li>使用自动洗板机交换用50μlPBS固定网上平台。注:样品现已准备在需要时从BSL3取出。 注意:从BSL3设施任何样品的去除受程序的风险评估和验证，并取决于对生物安全的有关法规。 </li></ol> 4.染色<ol><li>用50μl的PBS洗涤细胞两次。 </li><li>透于50μl0.1％的Triton-X-100的PBS细胞10分钟。 </li><li> W¯¯灰细胞三次，用PBS。加入20μl的PBS含有1微克/毫升的DAPI（4&#39;，6-二脒基-2-苯基）孵育在室温下30分钟。 </li><li>洗涤细胞三次在PBS避光染色用铝箔。 </li></ol> 5.成像<ol><li>设置用于图像采集的自动化宽视场显微镜。 注意:用于使用MetaXpress软件分子器件ImageXpress显微镜的设置如下所述。适当的硬件规格和图像处理软件替代2D宽视场显微镜设备可以使用（详见材料/设备表）。 <ol><li>选择10X客观，相机分级= 1，增益= 1。 </li><li>选择对应于该384孔板的板形式。 </li><li>获得每孔9点。 </li><li>使用"启用基于激光焦点"和"聚焦板井底"。 </li><li>设置"第一井"作为初始为好发现样品和站点自动对焦"所有网站"。 </li><li>使用DAPI通道手动设置Z-偏移焦点。 </li><li>手动选择Z-从偏移量为DAPI所有其他通道。 </li><li>手动修正的曝光时间的所有信道，以确保一个较宽的动态范围，低曝光过度。 <ol><li>获取使用自动曝光功能的代表部位的图像。使用此曝光时间图像3-5遗迹遍布板及以上的所有站点手动调整曝光时间，直到亮的像素达到了〜最大亮度的80％。 </li></ol></li><li>图片使用端点检测的DAPI和GFP渠道全板。对于进入试验选择另外RFP通道。 </li></ol></li></ol> 6.自动图像分析注:CellProfiler 2 月15日被用作图像分析软件来分割细胞和细菌的对象并执行自动测量电所确定的对象中对此语句。该软件提供了单独的模块，其可以被组合成一个管道，将连续执行上的所有图像的模块图像分析算法，以自动执行特定的图像分析的任务。按照该协议，安装CellProfiler 2.1.1或更高版本。然后，加载提供管道，并按照以下说明调整模块中所需的参数。所有管线的各个模块的描述可以在补充文件中找到。 注意:两个单独的管道被用于每个测定。第一管线计算阴影模型，其用于通过所述第二管道以校正之前分析图像。阴影校正被施加到图像以减少从显微镜不均匀光路的影响。计算在所述第一管道中的阴影模型是一个漫长的过程，但结果会更高的准确性。 <ol> &lt;LI&gt;端点检测<ol><li>计算着色模型<ol><li>进入"文件"菜单，选择"导入"→"从文件管道..."，然后选择所提供的管道"BrucellaShadingCorrectionPart1-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和所产生的着色模型输出文件夹。 </li><li>在模块（1）"LoadImages"，调整的名称为"文字，这些图像具有在共同的"相匹配的图像的名称。 </li><li>确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始着色模型的计算。 </LI&gt; </ol></li><li>运行图像分析<ol><li>进入"文件"菜单，选择"导入"→"从文件管道..."，然后选择所提供的管道"BrucellaEndpointWithShadingCorrectionPart2-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和生成的电子表格输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>在模块（2）"LoadSingleImage"，通过选择的位置，并在两个着色模型的文件名加载6.1.1下计算出的着色模型。 </li><li>在模块（4） - （5）"ImageMath"，调整参数相匹配的显微摄像机的位深度"通过乘以第一图像"。为8位和16位图片参数设置为"1.0"，12位图像参数设置为"16.0"。 </li><li>在模块（9）"ImageMath"调整参数"通过第二图像正片叠底"的值（开始与0.25），这将抑制DAPI染色布鲁氏菌信号，而抑制细胞核。 <ol><li>点击"开始测试模式"，然后激活通过点击模块的相应的图标暂停符号和模块（9）显示功能。 注:暂停符号会显示为黄色，而如果激活眼部符号会显示一个开放的眼睛。 </li><li>点击"运行"运行分析到与主动暂停符号模块（9）。要为模块，按"步"测试值。 </li><li>右键单击新开张的形象，并设置"图像对比度"到"日志归一化"。多次回踩模块（9），调整参数"乘以第二形象"，以及步过Ť他再次模块，直至而在指示过减法的同时黑色区域，结果图像中最小的布鲁氏菌信号被完全抑制。 </li></ol></li><li>在模块（10）"IdentifyPrimaryObjects"，设置参数"下限门槛"足够高（从零开始），使原子核是分段和背景被忽略。 <ol><li>以在模块为模块（10），步骤识别物有所值并如步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到核的良好分割为显示的输出图像中实现。 注:设置超过背景强度更高的阈值是空的网站尤其重要。 </li></ol></li><li>在模块（15）4; FilterObjects"，调整参数"最小值"，以便在核细胞清晰可见布鲁保持，和所有其他被滤除在此步骤中，忽略布鲁细胞核外。 <ol><li>要确定一个很好的价值，步过该模块，如步骤6.1.2.6.1和6.1.2.6.2说明显示输出图像。从零，其中标识为受感染的所有单元格开始，并增加小步骤的值，直到仅在核细胞清晰可见布鲁氏菌被保留。 </li></ol></li><li>在模块（16），"FilterObjects"，调整参数"最小值"，使细胞清晰可见布鲁氏菌在核周被保持，并且过滤掉所有其他细胞。使用类似的方法与先前模块中。 </li><li>在模块（17）"FilterObjects"，调整参数"最小值"，使细胞清晰可见布鲁氏菌在涡onoi胞体被保留，并过滤掉所有其他细胞。使用类似的方法与先前模块中。 注意:Voronoi单元体是核的径向延伸由25个像素与相邻的Voronoi细胞体没有重叠。 </li><li>退出测试模式，并确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始分析。 </li><li>当分析完成后，检查所得到的PNG图像以及该CSV片，以确保分析整个板可靠地工作。 </li></ol></li></ol></li><li>进入分析<ol><li>计算着色模型<ol><li>进入"文件"菜单，选择"导入"→"管道从文件..."，然后选择提供Ð管道"BrucellaShadingCorrectionPart1-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和所产生的着色模型输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>确保没有模块取消选中旁边模块的所有符号眼球运行完整分析前展示自己的显示器。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始着色模型的计算。 </li></ol></li><li>运行图像分析<ol><li>进入"文件"菜单，选择"导入"→"管道从文件..."，然后选择providED管道"BrucellaEntryWithShadingCorrectionPart2-Ver007"。当被问及是否将传统的管道进行转换，回答"不转换"。 </li><li>进入"输出"→"查看输出设置"，然后选择与图像输入文件夹和生成的电子表格输出文件夹。 </li><li>在模块（1）"LoadImages"调节"的文本，这些图像具有在共同的"相匹配的图像的名称的名称。 </li><li>在模块（2）"LoadSingleImage"，通过选择的位置，并在两个着色模型的文件名加载6.2.1下计算出的着色模型。 </li><li>在模块（4） - （5）"ImageMath"，调整参数相匹配的显微摄像机的位深度"通过乘以第一图像"。对于8位和16位的图像参数设置为"1.0"，12位图像参数设置为"16.0"。 </li><li>在模块（6）"IdentifyPrimaryObjects"，设置参数"罗WER阈值"不够高的约束，只有原子核是分段和背景被忽略。 <ol><li>以识别模块（6），步骤在模块良好的值，并在步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到核的良好分割为显示的输出图像中实现。 注意:设置上述背景的阈值是空的网站很重要的。 </li></ol></li><li>在模块（8）"IdentifyPrimaryObjects"，设置参数"下限阈值"不够高，只有布鲁氏菌是分段和背景被忽略。 <ol><li>以识别模块（8），步骤在模块良好的值，并在步骤6.1.2.6.1和6.1.2.6.2说明显示输入图像。 </li><li>右键单击输入图像和显示直方图。 注意:直方图的峰通常表示背景。 </li><li>从背景强度开始增加参数，直到所显示的输出图像中的布鲁氏菌的良好的分割实现。 注意:设置上述背景的阈值是空的网站很重要的。 </li></ol></li><li>在模块（11）"FilterObjects"，调整参数"最小值"，使背景和文物被过滤掉的，只有殖民地布鲁氏菌保持。 <ol><li>要确定一个很好的价值，步过该模块，如步骤6.1.2.6.1和6.1.2.6.2说明显示输出图像。在6.2.2.7和逐步选择的值开始增加，直到仅布鲁氏菌的对象保持。 </li></ol></li><li>退出测试模式，通过unche运行完整分析之前，确保没有模块的展示自己的显示器盛泰旁边模块的所有眼球的符号。 注意:此显著减小所需的计算资源，同时处理管道。 </li><li>按"分析图像"开始分析。 </li><li>当分析完成后，检查所得到的PNG图像以及该CSV片，以确保分析整个板行​​之有效。 </li></ol></li></ol></li></ol> 7.感染评分注意:这对细胞活力显著影响的siRNA必须谨慎考虑，因为这可以促进假阳性发现。一个改变的细胞数会影响实际MOI和必需基因靶向可以对病原体感染多效性影响。而通过的siRNA不完全耗尽允许必需基因的研究中，这些目标必须被通过其他方法（ 例如，药物干扰），以证实它们作为宿主因素的作用进行验证感染期间。 <ol><li>端点分析<ol><li>打开步骤6.1.2.12产生该CSV片和除以感染的细胞的数目（CellProfiler读出:"Count_InfectedCells"）计算出每孔的感染速率由细胞的总数（CellProfiler读出:"CountNuclei"）。如果每口井多个站点已经成像，首先总结所有站点每口井，建成了每孔数感染的细胞和细胞每孔总数。 </li><li>执行ž得分正常化占板与板的变化，如果板含有足够的非击中的siRNA。假设有足够数量每盘不打siRNA的中频饱满库筛选。 <img alt="式（1）" src="/files/ftp_upload/54263/54263eq1.jpg" /></li></ol></li><li>进入分析<ol><li>打开步骤6.2.2.10产生该CSV片和除以感染的细胞的数目计算出每孔的感染率（CellProfiler读出:"Count_InfectedCeLLS"）的细胞总数（CellProfiler读出:"CountNuclei"）如果每口井多个站点已经成像，首先总结所有站点每口井，建成了每孔数感染细胞和每孔总数量的细胞。 </li><li>量化感染细胞的细菌载荷，估计由每个感染细胞内细菌所占据的平均面积。为此，除所有细胞内细菌与细胞重叠的累计面积（CellProfiler读出:"AreaOccupied_AreaOccupied_TrueIntPathogenInCells"）由被感染的细胞在此站点的数目。考虑到伪影，使用该读出作为阱读出的所有地点的中位数。 注意:如果该试验被用作含大量参与布鲁氏菌感染基因的跟进检测，不适用Z分数正常化。相反，如果使用模拟井作为参考，以占板与板的变化进行正常化。 </li></ol></li></ol>

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The authors declare no conflicts of interest.

Bacterial pathogens have evolved numerous strategies to manipulate eukaryotic host cells to their benefit. Pathogens causing acute infections often show rapid proliferation which is accompanied by significant alarming of the immune system and loss of viability of infected cells. In contrast, Brucella and other pathogens that cause chronic infections manage to establish long-lasting interactions within host cells. Therefore, bacteria need to fine tune host cell functions to their benefit without disrupting cellul...

布鲁氏菌物种是属于类α-变形菌的革兰氏阴性，兼性细胞内病原体。它们引起流产和不孕在其自然宿主如牛，山羊，绵羊或导致在流行地区严重的经济损失。布鲁氏菌病是最重要的人畜共患病引起全球每年超过五十万新的人类感染1之一。传输给人类最常用的是与受感染动物的直接接触或通过污染的食物摄入引起的，如未经高温消毒的牛奶。发热疾病的症状是非特异性的，这会导致困难布鲁氏菌病诊断。如果不治疗，患者可发展为慢性感染更严重的症状，如关节炎，心内膜炎，和神经病2。 细胞水平上的布鲁氏菌能够侵入吞噬细胞和非吞噬细胞和细胞内内复制车厢被称为布鲁氏菌含泡（BCV）。细菌的内化外消旋，卢，和Cdc42 3直接激活需要肌动蛋白骨架重排。真核宿主细胞内部，沿BCV内吞途径贩卖，尽管与溶酶体的相互作用，细菌设法避免降解4。由囊泡ATP酶的BCV酸化时需要引起细菌IV型分泌系统（T4SS）5的表达。据认为，由T4SS分泌细菌效应是必不可少的布鲁氏菌 ，以确立其复制利基，由于T4SS 6的缺失或囊泡ATP酶引线抑制在建立细胞小生7的缺陷。细菌中，直到他们贩卖的阶段不复制 达成ER衍生的液泡车厢8。一旦细胞内增殖时，BCV在CL发现与ER标志物如钙联接蛋白和葡萄糖-6-磷酸酶6 OSE关联。 由布鲁氏菌进入细胞，避免溶酶体降解，最后复制在ER-状隔间的分子机制仍是未知。参与感染的不同步骤宿主因素主要被确定的目标的方法或小规模小干扰RNA（siRNA）在果蝇细胞9执行屏幕。这些都揭示个人的宿主因素的布鲁氏菌感染过程中的贡献，但我们仍远未整个过程有全面的了解。 这里，协议，允许使用与自动宽视场荧光显微镜和自动图像分析相结合的大型RNA干扰（RNAi）筛选人类宿主因子的鉴定呈现。 HeLa细胞反向siRNA转染作为describ进行ED早期10,11稍作修改。端点测定覆盖除了出口和感染邻近细胞的布鲁氏菌细胞内的生命周期的很大一部分。为了进一步表征在端点测定法鉴定的命中，则使用一个修改协议，以确定所涉及的感染的早期步骤的因素。 组成型表达GFP一个流产布鲁氏菌菌株用于细菌的地方允许感染细胞两天端点检测。在此期间，细菌进入细胞，投放到ER衍生的复制利基，并在周围核空间复制。高水平的GFP信号然后可用于可靠地检测含复制的细菌的单个细胞。 为了研究在高通量检测布鲁项，它是能够细胞内外细菌区分重要。该方法在这里circumv介绍经济需求测试的细胞内和细胞外的细菌差速器抗体染色。它是基于一个布氏杆菌菌株表达结合的四环素诱导的GFP与DsRed的组成型表达。组成型DsRed的标志物的存在允许在样品中存在的所有的细菌的鉴定。 GFP表达是由庆大霉素加入无毒四环素类似物无水四环素（ATC）与细胞外细菌的灭活同时的（GM）诱导。而细胞不透抗生素了Gm杀死胞外细菌，的aTc可以进入宿主细胞并在细胞内细菌选择性诱导GFP表达。此双报告允许使用宽视场显微镜从胞菌（仅DsRed的信号）单胞菌（GFP和DsRed的信号）的健壮的分离。为了通过细胞内布鲁氏菌达到可检测的GFP表达，我们已经发现，通过的aTc 4小时的诱导导致RELI能信号。类似的诱导方案在胞内细菌选择性地表达GFP先前已用于研究细胞内的志贺氏菌 12。

<table><tbody><tr><td>Tryptic Soy Agar (TSA)</td><td>BD</td><td>236950</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Fluka</td><td>22092</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Skim milk</td><td></td><td></td><td></td></tr><tr><td>250 ml screw cap bottle</td><td>Corning</td><td>8396</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Gibco</td><td>10270</td><td>Heat-inactivated</td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>Gibco</td><td>15400-054</td><td>10x stock solution, dilute 1:10 in PBS</td></tr><tr><td>Scepte 2.0 Cell Counter</td><td>Merck Milipore</td><td>PHCC20060</td><td>Alternative cell counting devices can be used</td></tr><tr><td>Greiner CELLSTAR 384-well plate</td><td>Sigma-Aldrich</td><td>M2062</td><td></td></tr><tr><td>Peelable aluminum foil</td><td>Costar</td><td>6570</td><td></td></tr><tr><td>Reagent dispenser: &quot;Multidrop 384 Reagent Dispenser&quot;</td><td>Thermo Scientific</td><td>5840150</td><td>Alternative reagent dispenser can be used.</td></tr><tr><td>Transfection reagent: &quot;Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778-150</td><td></td></tr><tr><td>Automated plate washer: &quot;Plate washer ELx50-16&quot;</td><td>BioTek</td><td>ELX5016</td><td>This plate washer contains a 16-channel manifold suitable for 384-well plates. It fits into a biosafety cabinet and has a lid covering the plate during washing which reduces the risk of aerosol production. Alternative plate washers with similar features could be used.</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>Anhydrotetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>37919</td><td>100 &amp;#956;g/ml solution in 100% ethanol is kept at -20 &amp;deg;C protected from light in aluminum-foil</td></tr><tr><td>PBS</td><td>Gibco</td><td>20012</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20 &amp;deg;C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed.</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>Scrambled siRNA</td><td>Dharmacon</td><td>D-001810-10</td><td></td></tr><tr><td>Kif11 siRNA</td><td>Dharmacon</td><td>L-003317-00</td><td></td></tr><tr><td>ARPC3 siRNA</td><td>Dharmacon</td><td>L-005284-00</td><td></td></tr><tr><td>&lt;em&gt;Brucella abortus 2308&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>pJC43 (&lt;em&gt;apHT::GFP&lt;/em&gt;)</td><td></td><td></td><td>Celli &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;12&lt;/sup&gt;</td></tr><tr><td>pAC042.08 (&lt;em&gt;apht::dsRed, tetO::tetR-GFP&lt;/em&gt;)</td><td></td><td></td><td>Construction: pJC44 &lt;sup&gt;4&lt;/sup&gt; was digested with EcoRI followed by generation of blunt ends with Klenov enzyme and subsequent digestion with SalI. TetR-GFP was amplified from pNF106 using primer prAC090 and prAC092. Following digestion with SalI, the TetR-GFP product was ligated to the digested pJC44 vector.</td></tr><tr><td>Primer prAC090</td><td>Sigma-Aldrich</td><td></td><td>TTT TTG AAT TCT GGC AAT TCC GAC GTC TAA GAA ACC</td></tr><tr><td>Primer prAC092</td><td>Sigma-Aldrich</td><td></td><td>TTT TTG TCG ACT TTG TCC TAC TCA GGA GAG CGT TC</td></tr><tr><td>HeLa CCL-2</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>ImageXpress Micro</td><td>Molecular Devices&amp;nbsp;</td><td>IXM IMAGING MSCOPE</td><td>Automated cellular imaging microscope equipped with a precision motorized Z-stage. Alternative systems for automated microscopy and alternative components for hard- and software specified below can be employed.</td></tr><tr><td>High-Speed Laser Auto-Focus</td><td>Molecular Devices&amp;nbsp;</td><td>1-2300-1037</td><td></td></tr><tr><td>CFI Super Fluor 10X objective</td><td>Nikon&amp;nbsp;</td><td>MRF00100</td><td>N.A 0.50, W.D 1.20 mm, DIC Prism: 10X, Spring loaded</td></tr><tr><td>Photometrics CoolSNAP HQ Monochrome CCD Camera</td><td>Molecular Devices&amp;nbsp;</td><td>1-2300-1060</td><td>1,392 x 1,040 imaging pixels, 6.45 x 6.45 &amp;micro;m pixels, 12 bits digitization</td></tr><tr><td>MetaXpress software</td><td>Molecular Devices&amp;nbsp;</td><td>9500-0100</td><td></td></tr><tr><td>LUI-Spectra-X-7</td><td>Lumencor</td><td>SPECTRA X V-XXX-YZ</td><td>Light engine. The following light sources are used: violet (DAPI), cyan (GFP), green/yellow (RFP)</td></tr><tr><td>Single Band Exciter for DAPI</td><td>Semrock</td><td>FF01-377/50-25</td><td></td></tr><tr><td>Single Band Emitter for DAPI</td><td>Semrock</td><td>FF02-447/60-25</td><td></td></tr><tr><td>Single Band Dichroic for DAPI</td><td>Semrock</td><td>FF409-Di03-25x36</td><td></td></tr><tr><td>Single Band Exciter for GFP</td><td>Semrock</td><td>FF02-472/30-25</td><td></td></tr><tr><td>Single Band Emitter for GFP</td><td>Semrock</td><td>FF01-520/35-25</td><td></td></tr><tr><td>Single Band Dichroic for GFP</td><td>Semrock</td><td>FF495-Di03-25x36</td><td></td></tr><tr><td>Single Band Exciter for RFP</td><td>Semrock</td><td>FF01-562/40-25</td><td></td></tr><tr><td>Single Band Emitter for RFP</td><td>Semrock</td><td>FF01-624/40-25</td><td></td></tr><tr><td>Single Band Dichroic for RFP</td><td>Semrock</td><td>FF593-Di03-25x36</td><td></td></tr></tbody></table>

microscopy-based assays for high-throughput screening of host factors involved in brucella infection of hela cells

布鲁氏菌的物种是感染的动物作为天然宿主兼细胞内病原体。传播给人类最常通过与受感染的动物的直接接触或通过污染的食物摄入引起的，并可能导致严重的慢性感染。 布鲁氏菌可以侵入的专业和非专业的吞噬细胞和内质网（ER）衍生空泡内复制。 布鲁氏菌对进入宿主细胞，溶酶体降解的回避和复制在ER形腔室所需的宿主因素仍是未知。在这里，我们描述两个测定以鉴定参与在HeLa细胞布鲁氏条目和复制宿主因素。该协议描述了使用RNA干扰的同时，可应用替代的筛选方法。该测定法基于检测荧光标记的细菌使用自动化在荧光标记的宿主细胞宽视野显微镜。荧光图像用CellProfiler标准化图像分析管道，允许单细胞为基础的感染得分进行分析。 在端点检测，细胞内的复制感染两天后进行测量。这使得细菌交通到增殖的细菌进入。 布鲁氏菌已成功建立了一种细胞内的利基将因此已在宿主细胞内增殖强烈围绕后12小时开始他们的复制利基。因为细胞内的细菌会的数量大大超过个别的细胞外或细胞内非复制性的细菌，应变组成型表达绿色荧光蛋白可以使用。然后强GFP信号是用来识别被感染的细胞。 与此相反，对于条目测定是必不可少的细胞内和细胞外的细菌区分开来。这里，被用于四环素诱导的GFP的菌株的编码。感应与由庆大霉素胞菌的同时失活的GFP使细胞内和细胞基于所述GFP信号外细菌之间的分化，与仅细胞内细菌能够表达GFP。这允许鲁棒检测单胞菌的启动细胞内增殖之前。

图1A示出用于自动识别在端点检测感染细胞的图像分析的一个例子。用DAPI染色的HeLa细胞的细胞核被确定，包围核8像素宽度的周围核，以及由25个像素的核的扩展Voronoi单元体进行了计算。因为细菌主要增殖在围核的空间，在这个范围内的小区的GFP强度是最稳健的测​​定感染的和非感染的细胞之间进行区分。在某些情况下，细菌被发现围核或覆盖的外扩散与?...

Watch this Scientific Journal Video about Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells at JoVE.com

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

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

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

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

microscopy-based-assays-for-high-throughput-screening-host-factors

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.2K 次观看.  University of Basel. Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

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

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

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

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

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

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 ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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 ...

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 ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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

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

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

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

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

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

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 High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

在参与宿主因素的高通量筛选基于显微镜的检测方法

摘要

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此视频中的章节

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

单细胞测量液泡破裂引起细胞内病原体

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

生成和多表型高内涵筛选

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

系统抑制剂靶向细胞内的疗效和毒性筛选

高通量兼容检测，以评估对巨噬细胞传代药物疗效

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

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究