PS是比尔和梅琳达·盖茨大挑战奖牌OPP1141322的收件人。 PV由瑞士国家科学基金会早期博士后移动奖学金和Roux-Cantarini博士后研究金支持。 PJS由HHMI授权和ERC-2013-ADG 339579解密支持。

1.细菌和宿主细胞的制备<ol><li>使用无菌镊子分别在24孔或12孔板中转移12或14毫米玻璃盖，每个实验条件计数一个孔。在组织培养罩中通过UV处理灭菌15分钟。 注意:包括用于细菌攻击的控制井和通过外源胁迫进行SG诱导。 </li><li>对于具有12mm盖玻片的24孔板，将1×10 5个哺乳动物细胞（ 例如 HeLa细胞）种在盖玻片上;用无菌吸头向下压盖玻片。让细胞在37℃下在适合每种细胞类型的培养基中的5％CO 2组织培养培养箱中过夜。 注意:板细胞使细胞的汇合在第二天之间为40-60％之间。 <ol><li>对于HeLa细胞，在含有非必需氨基酸（NEAA）和10％补充的胎牛血清（FCS）的Dulbecco&#39;s Modified Eagle培养基（DMEM）中孵育。通过在56℃水浴中加热30分钟来补充FCS，15分钟后将血清旋转一次。 </li></ol></li><li>为了划破细菌，使用无菌移液器吸头从储存在-80°C冰箱中的细菌甘油储备液（20％甘油）中除去少量，并将其放在标记的板上。使用无菌环将细菌扩散到板的约10％。使用新的无菌环并将其拖曳通过涂片，使3条平行线，半个长的板。穿过这些线穿过板的其余部分。在37℃孵育板O / N。 <ol><li>从新鲜的条纹板中选择一个细菌菌落（ 例如S. flexneri ）。避免使用长于1周的细菌菌落。 </li><li>对于S. flexneri菌株M90T，将来自新鲜条纹的胰蛋白酶大豆琼脂-0.1％刚果红琼脂平板的红色菌落接种到15mL管中的8mL胰蛋白酶大豆肉汤中;拧紧盖子，并在30℃以222转/分的速度孵育振荡6℃。 小心:不要使用大的白色菌落，因为它们已经失去了毒力质粒并且是非感染性的。 </li></ol></li></ol>宿主细胞的细菌挑战<ol><li> 准备细菌以最大限度地发挥感染潜力。 <ol><li>对于S. flexneri ，传代培养细菌通过向8mL胰蛋白酶大豆琼脂中加入150μLO / N培养物，并在37℃下孵育222rpm至晚期指数期（〜2h）以诱导毒力基因表达并增强感染。 <ol><li>当培养物的光密度为0.6至0.9（在600nm处测量）时，将1mL培养物转移到1.5mL管中。颗粒细菌在8,000×g下2分钟，并在OD 600 = 1时重悬于感染培养基（含有NEAA的DMEM，缺少FCS））。因此，如果OD 600为0.85，则重悬于850μL。 注意:对于S. flexneri，在OD 600 = 1时，每mL时将有8×10 8个细菌在8 mL培养基中培养。感染的多重性（MOI）为20是合适的。对于其他病原体，OD 600处的细菌数量和适当的MOI需要根据经验确定。 </li></ol></li><li>为了增强细菌与宿主的相互作用，用聚-L-赖氨酸（PLL）包被细菌。 注意:灵芝的PLL处理对SG动力学没有影响。需要评估其他病原体对于PLL治疗的适应性。 <ol><li>为了用PLL包被细菌，以8,000×g离心1mL细菌培养物2分钟，然后以OD 600 = 1在磷酸盐缓冲盐水（PBS）中将沉淀重悬于10μg/ mL的PLL中。 </li><li>将细菌溶液加入一个小盘，如12孔板中的一个孔中，并在平板振荡器上轻轻搅拌，在室温（〜20℃）下孵育10分钟。 </li><li>颗粒细菌在8,000 xg 2分钟，去除多余的PLL。将沉淀重悬于1 mL PBS中d重复这个洗涤步骤两次。最终洗涤后，重新悬浮于感染培养基OD 600 = 1。 注意:这里， S. flexneri的感染培养基是具有20mM HEPES w / o FCS的宿主细胞培养基。 </li></ol></li></ol></li><li> 准备细胞进行细菌攻击。 <ol><li>使用抽吸泵从哺乳动物HeLa细胞中除去培养基。加入500μLRT HeLa细胞培养基以洗涤细胞，旋转，使用抽吸泵除去培养基，并用500μLRT适当的感染培养基（ 例如 ，具有20mM HEPES的FCS的宿主细胞培养基）替代。 </li><li>注意:对于所有洗涤步骤，快速工作，一次只处理最多4个盖玻片，以避免细胞干燥。 </li></ol></li><li> 挑战准备HeLa细胞与细菌感染20-50％的细胞。 注意:每个细菌种类需要确定适当的时间和MOI。一般来说，一个好的开始是37分30分钟°C，MOI为10。 <ol><li>对于S. flexneri，使用两种方法之一（步骤2.3.1.1或2.3.1.2）攻击HeLa细胞细胞。 <ol><li>将非PLL处理的细菌（20μL的OD 600 =500μL培养基中的24孔板的1孔）在13000xg下在细胞上10分钟。 </li><li>在室温下，加入PLL包被的细菌（15μL的OD 600 = 24孔板在500μL培养基中的1孔）15分钟，以使细菌沉降到细胞上。 </li></ol></li><li>通过将沉降细菌的细胞置于37℃的组织培养箱中30分钟使感染发生。 注意:对于短时间点，通过将板放置在37°C水浴中15分钟而不是在组织培养箱中同步S. flexneri感染。 </li></ol></li><li> 通过洗涤细胞停止感染。 <ol><li>为了洗涤细胞并除去多余的细菌，使用抽吸泵除去培养基，加入1 mL新鲜预热（37℃）HeLa培养基（DMEM，10％FCS，NEAA）。旋转培养基，取出并更换新鲜培养基。重复两次，并在细胞上留下新鲜的培养基。 <ol><li>对于S. flexneri或其他细胞内细菌，在感染HeLa细胞和洗涤后，用含有50μg/ mL庆大霉素的标准组织培养基代替培养基以杀死细胞外细菌并防止进一步的细胞侵袭。 注意:不要在细胞外细菌的培养基中加入抗生素。 </li></ol></li></ol></li><li>在细胞培养箱中孵育细胞达所需时间。 注意:对于S. flexneri ，感染可能长达6小时，具体取决于细胞类型。对于HeLa细胞，使感染进行1.5-2小时，然后加入外源胁迫以诱导SG形成。对于Caco-2感染，其中志贺氏菌传播细胞对细胞，感染30分钟至6小时，然后加入外源性应激。策在这一点上，lls可以固定，而不增加外生应激来评估由于细菌感染而形成的SG（在这种情况下，进行到第4部分）。 </li></ol>通过添加外源性应激诱导应激颗粒形成<ol><li>使用抽吸泵将培养基从感染和未感染的HeLa细胞中取出，用或不加压力培养基替换含有500μL合适培养基的培养基。 注意:应激诱导条件包括以下内容（见下文）。对于额外的治疗，参见Kedersha 等 13 。 <ol><li>对于热休克治疗，使用含20mM HEPES的常规培养基，并将板置于44℃水浴中30分钟。 </li><li>对于克霉唑（诱导线粒体应激）治疗，使用无FCS的常规培养基与20μM克霉唑，并在组织培养箱中孵育1小时。 注意:存储20米的一次性等分试样M克霉唑在二甲基亚砜（DMSO）中的溶液在-20℃下长达4个月。使用DMSO载体作为对照细胞。 </li><li>对于亚砷酸盐（诱导氧化应激）治疗，使用0.5μM亚砷酸盐的常规培养基，并在组织培养箱中孵育1小时。 注意:将一次使用0.5mM亚硫酸钠的等分试样储存在-20°C长达6个月。 小心:克霉唑和亚砷酸盐对环境是有害的和危险的，必须妥善处置。 </li><li>对于去甲基脱氨基（DMDA） - 胺处理（抑制真核生物翻译起始），使用具有50-200nM DMDA-百日咳胺的常规培养基，并在组织培养箱中孵育1小时。 注意:将DMDA-pateamine储存在-80°C。将试剂储存为小量试样以避免冻融。 </li></ol></li></ol> 4.应力颗粒形成的固定和免疫荧光分析注意:处理控件和实验盖玻片同时避免染色差异，可能会影响后续步骤中的图像分析。对照样品不包括感染和无SG诱导治疗，感染样本有和没有SG诱导治疗。 <ol><li>使用抽吸泵完全取出培养基，并通过在PBS中加入0.5mL RT 4％多聚甲醛（PFA）来固定样品。在室温下孵育30分钟。 小心:PFA对处理器和环境有害。采取适当措施保护处理者并处理化学废物。 注意:或者，修复15分钟，然后用-20°C预烧的甲醇替换10分钟，以保留更多的SG标记13的细胞质定位。 </li><li>用移液器取出PFA，并将液体置于适当的废物容器中。用1mL Tris缓冲盐水（TBS:25mM Tris，pH 7.3; 15mM NaCl）洗涤盖玻片。孵化盖玻片2分钟，轻轻晃动在洗涤过程中的平板振动筛上。 </li><li>使用抽吸泵完全清除TBS，并在TBS中加入500μL0.3％Triton-X100 10分钟以使细胞透化。 </li><li>用抽吸泵去除透化溶液。用1mL TBS替代，轻轻旋转板，抽吸除去TBS，并在室温下加入1mL封闭溶液（TBS中5％FCS）1小时。 </li><li>在TBS中的5％FCS中制备初级抗体溶液（1:300稀释）。计算每12 mm盖玻片50μL，并为所有盖玻片一次充足。 注意:使用常见的一级抗体为G3BP1，eIF3b和TIA1。 </li><li>将50μL的一级抗体混合物固定在塑料石蜡膜（石蜡膜）上，将其牢固地粘贴到台面或室上。 注意: 材料表中提供了标准SG标记的列表，但SG的组成可能取决于施加的应力。其他SG标记列在Kedersha 等人 13 </sup&gt;许多SG标记物的灵敏感染的预期结果列于表1中 。 </li><li>用镊子拿起盖玻片，将盖玻片的边缘沾到组织上，短暂释放镊子以清除多余的液体，轻轻地将面朝下放在滴下。孵育盖玻片在室温下1.5小时或在4℃在潮湿室中过夜。 注意:为了准备一个潮湿的室，将预润湿的组织沿着一个牢固封闭的房间的边缘，内衬塑料石蜡膜。 </li><li>将每个盖玻片用镊子转移到装有1mL TBS的新的24孔板的孔中;在平板摇床上温和摇动孵育5分钟。使用抽吸泵吸取每个井中的TBS，更换为1 mL TBS，放回平板振荡器5 min。重复洗一次。 注意:通过用纸巾去除多余的液体并用水清洗表面，重新使用石蜡膜。在使用前确保石蜡膜完全干燥g用于随后的染色步骤。 </li><li>在TBS中制备二抗溶液（稀释1:500-1:2000）。为了染色细胞核和细菌，向混合物中加入2μg/ mL DAPI。为了染色肌动蛋白，加入荧光鬼笔环肽。将50μL二抗混合物吸取到塑料石蜡膜上。 注意:当使用G3BP1（山羊抗体）时，推荐使用高度纯化的交叉吸收驴二抗来进行不同SG标记的共定位研究。选择具有非重叠光谱的荧光标记的二抗。 eIF3b清楚地染色细胞的细胞质，因此是描绘细胞的良好标记。肌动蛋白染色进一步有助于细胞间细胞接触部位和细菌进入区域的细胞。 </li><li>用镊子拿起盖玻片，将盖玻片的边缘沾到组织上，短暂释放镊子，以清除多余的液体。轻轻地将盖玻片面朝下放在滴下并孵化盖玻片在黑暗中在室温下1小时。 </li><li>使用镊子将盖玻片放回装满新鲜1 mL PBS的24孔板中。确保盖玻片被PBS完全覆盖。洗涤细胞3次，轻轻晃动5分钟。 注意:在二次抗体的荧光团对光敏感的情况下，将洗涤液中的板保持在盖子下以防止光照。 </li><li>将盖玻片简单地浸入去离子水中以除去盐，从边缘干燥到组织上，并将盖玻片安装在载玻片上的5μL荧光装载介质上。使用指甲油成像之前密封盖玻片。保持幻灯片在4°C短期（周）或-20°C长期（月）存储。 注意:密封时应避免气泡，否则会损害图像质量。 </li></ol>荧光成像注意:参考显微镜用户手册进行优化设置。 <ol><li>使用40或63X油浸透镜的共焦显微镜获取图像。选择所需的荧光通道进行图像采集。使用多个荧光通道时，选择顺序采集模式。 注意:从实验样品开始，其中已经诱导SG最强，以避免后续样品过度暴露。确保在整个细胞的整个深度不要有过度曝光的SG。 <ol><li>在显微镜设置中设置12或更高的位大小，以确保图像分析的准确性。 </li><li>设置图像堆叠以覆盖单元格的整个深度。检查不同的通道和不同的SG标记，以确保包含整个范围。将单元格的开头设置在单元格顶部的单元格顶部，并在单元格顶部的高度处，不再看到SG标记。 </li><li>获取堆栈。保持堆叠高度相同的所有图像。 注意:不要改变将用于后续比较的对照和实验样品之间的采集设置。 </li></ol></li></ol>图像分析注意:在这里，描述使用免费ICY的折叠堆栈的SG分析。 3D重建的图像分析也可以使用其他专门的软件来完成。 SG检测可以通过为该协议建立的完全自动化的工作流程（可从http://icy.bioimageanalysis.org/protocol/Stress_granule_detection_in_fluorescence_imaging获得）来执行。为了使用这个协议，核需要用DNA染色剂染色软件以找到每个细胞的中心，并且细胞边缘需要用细胞质标记（如eIF3b）或肌动蛋白染色来标记软件以识别细胞边界。自动工作流程可以用于验证手动派生的结果，或者直接用于高分辨率检测细胞边界时进行分析，这将是mostly取决于细胞的密度和用于检测细胞边界的标记。 <ol><li>使用ImageJ或Fiji，折叠图像堆栈（图像&gt;堆栈&gt; Z投影），并另存为.tiff文件。 注意:为每个图像创建一个新的文件夹，因为图像分析软件将其结果链接到原始图像的站点。 </li><li>将一个控制和实验的.tiff图像加载到图像分析平台（File&gt; Open）。转到Sequence窗口。在"查找表"中向下滚动到通道参数。 </li><li>通过单击所有通道选项卡的复选框来停用所有通道。单击通道0，然后单击上面的颜色条选择首选颜色。根据需要增加通道的强度，通过单击并移动强度区域中的线来查看所有结构。对所有渠道重复此操作。 注意:停用给定任务不需要的通道，以尽量减少样品分析中的偏差。 </li><li>滚动在"画布"选项卡中，通过调整缩放选项卡，放大每个字段大约10个单元格。 </li><li>使用多边形函数工具（在顶部栏中）来描绘图像中单元格的单元格边界。使用肌动蛋白染色剂或细胞溶质标记物进行细胞边界描绘（ 例如 eIF3b）。 <ol><li>单击"文件和ROI"工具中的多边形功能。通过单击单元格开始描绘，然后通过重复点击单元格边界来使锚点延伸。要完成投资回报率，请再次单击"文件和投资回报率"工具中的多边形函数。 注意:确定被感染细胞的亚种群。样品由感染细胞和未感染细胞组成。要识别细菌，请使用DAPI染色剂或荧光细菌（如表达GFP的细菌）。增加强度，以确保所有细菌都能看到。 </li><li>要命名ROI，请转到右侧的ROI窗口和click在图像中感兴趣的单元格。这将突出显示ROI选项卡中相应的ROI。双击ROI名称并修改名称（ 例如感染细胞＃1）。 注意:如果要使用一张图像来分析感染细胞和未感染细胞，可以将图像保存在两个不同的文件夹中，并分别描绘感染细胞和非感染细胞，生成两个不同的电子表格，这将有助于分析后来。 </li></ol></li><li>在"检测和跟踪"选项卡（顶部栏）中打开点检测器应用程序。在"输入"选项卡中，将选择当前图像。不要改变任何东西在"预处理"选项卡中，选择要分析的通道。 注意:在任何给定时间只能分析一个通道。如果使用完全自动化的分析序列，其参数已被检查以获得令人满意的结果，则可以选择批次分析。 <ol><li>在"检测器"选项卡中，选择"在黑暗背景下拍摄亮点"，然后选择适当的比例和灵敏度，通过在对照和实验样品中测试不同的设置和交叉检测点检测。 注意:对于分辨率为2,048 x 2,048，像素大小为0.12μm2的图像，灵敏度为25至100的2级阈值通常是适当的，但每个SG标记必须经验性测试。设置斑点检测，使得在未处理对照样品中未检测到斑点，而在具有SG的实验样品中，计数所有小而大的SG。 </li><li>在ROI选项卡中，选择建议的ROI序列。在"过滤"选项卡中，选择"不过滤"。在"输出"选项卡中，选择相应的输出。 注意:选择启用特定文件有助于保存不同的检测条件或不同的通道。选择导出二进制图像和原始图像与ROI和检测是helpful进行质量控制分析。 </li><li>选择"删除渲染为ROI的前一个点"。选择要保存文件的文件夹，方法是单击"无文件选择"按钮。在"显示"选项卡中，选择所需的选项。单击"开始检测"。 注意:将创建包含导出的成像选项的名称和位置的文件夹。这些图像将被覆盖每个新的条件测试。要保留图像，请重命名文件夹，以便创建新文件夹或将图像从保存文件夹传输到新文件夹。对于图像的质量控制，选择显示检测标记，检测ROI数量和ROI数量和名称是有帮助的。 </li></ol></li><li>使用生成的电子表格合并和保存不同参数的数据，以测试每个图像或条件。 注意:要分析的参数包括每个投资回报率的SG数量，SG表面积和SG最大值平均和最小强度。 </li><li>使用能够分析，绘制和确定统计学意义的分析程序传输数据并进行分析。 </li></ol>

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作者没有什么可以披露的。

本文概述的方案描述了在存在或不存在外源胁迫的情况下，非感染细胞和感染细胞溶质病原体柔毛霉的细胞中的SG的诱导，定位和分析。使用免费的成像软件，协议允许对SG形成进行精确的定性和定量分析，以鉴别和统计学地解决给定表型的差异。 在感染，SG感应和成像部件的协议中有几个关键步骤。对于感染，重要的是与宿主细胞的细菌相互作用，如本文中概述的方案的入侵，尽可能同步。当在感染过程中早期研究细菌攻击的影响时，这尤其重要。我们以前表明，一些表型，如蠕虫感染后的eIF3b定位在野生型早期受到干扰其他表型，如含有G3BP1的SG的聚集，在以后的时间点更受影响。因此，为了精确描述细菌攻击的时间效应，建议使用感染同步。值得注意的是，为了分析SG形成，细胞应该处于生长的指数期，因此加压时的细胞密度也是重要的参数。它还将SG分析限制为非汇合细胞感染模型。另外一个重要的方面是在处理成像样本和图像采集过程中，控制和实验样品的协同处理。对于免疫荧光处理，所有样品应在相同的时间和相同条件下（ 即 RT Vs.4 ℃）下用相同的抗体工作稀释液进行染色，同时在洗涤阶段同样照顾每个盖玻片，安装o盖玻片。这将确保可定量和定量分析的可比较的免疫荧光染色。安装介质的差异在图像采集过程中对样品的漂白有显着影响，因此应保持恒定。类似地，在分析中要进行比较的所有样品的图像采集应在一次会议中进行，并且具有相同的设置，以最小化由激光强度或样品设置引起的差异。 使用外源性应力时，要妥善储存试剂并经常制作新的工作原料很重要。延长期（例如，克霉唑≥4个月）导致药物效力降低，并会影响SG形成。在加入细胞之前，应将试剂加入培养基中;如果观察到对照细胞中SG形成的降低或SG的聚集出现异常，则应测试试剂的ac应该制作新的股票。 一个限制是当存在太多的背景荧光时，使用斑点检测器的SG识别变得不太可靠。虽然这对于许多SG标记物来说不是问题，但是在SG诱导和非诱导条件下，一些例如eIF3b是清楚的细胞溶质标记物， 如图3所示更难分析。另外，对于每个SG标记，需要根据经验确定尺度和敏感度。另一个限制是，通过图像分析软件的分析最好使用2D图像，因此在光学切片或折叠堆叠上工作良好，然而这导致3D信息的丢失。因此，z投影的分析往往夸大了SG的规模，低估了SG的数量。如果认为对于特定表型是必要的，则可以用Imaris进行SG的3D分析以给出更精确的空间表征。 </p&gt; 可以使用图像分析软件的自动化点检测器，以标准化的方式快速执行SG表征。相比之下，手动方法来识别和描述SG，如先前所做的那样4 ，使分析开放更多的偏见，并且相当耗费时间。使用斑点检测器的SG分析也可以通过选择不同的灵敏度和尺寸阈值来定制以包括或排除小的SG聚集体，从而允许评估SG形成的不同方面的灵活性。 SG分析也可以使用已建立的自动化工作流程以完全自动的方式执行3 。在该工作流程中，通过DAPI染色鉴定细胞的中心，然后该程序允许可延展的球体向外生长以基于细胞质染色体（例如eIF3b）或肌动蛋白来描绘细胞边界。同时，使用半自动分析手动描绘单个细胞进行分析时，当细胞在簇中生长并且细胞边界难以自动化程序清楚地描绘时，这是有利的。在这些情况下，手动定义单元格边界可以消除假的正值或负值。 方案可适用于其他SG诱导条件和其他病原体，包括病毒，细菌，酵母和原生动物。特别感兴趣的可能是其他细胞溶质病原体，如立克次 氏 体菌， 土拉弗朗西斯菌 ， 假单胞菌和李斯特菌。对于每种感染因子，也可能是不同的细胞系，感染方案将不得不进行调整，并且经验确定外源性应激的样本时间。此外，使用图像分析软件的SG表征也可以在将来被扩展到活细胞成像。必须进行实时SG分析表示一个或多个荧光标记的SG标记在宿主细胞中的表达，从而允许在不同实验条件下解决SG形成的时间和特殊动力学问题。然后需要荧光标记的细菌来补充感染和未感染细胞中的实时SG分析。

在细胞水平上可视化宿主 - 病原体相互作用是获得对致病性策略和识别关键细胞途径的见解的有力方法。事实上，病原体可以用作确定重要细胞靶点或结构的工具，因为病原体已经发展为颠覆中枢细胞过程，作为其自身存活或繁殖的策略。可以通过重组表达荧光标记的宿主蛋白来实现细胞成分的可视化。虽然这允许进行实时分析，但具有特异标记宿主蛋白质的细胞系的产生是非常费力的并且可能导致不期望的副作用。使用特异性抗体检测细胞因子更方便，因为可以同时分析多个宿主因子，并且不限于特定的细胞类型。缺点是只有静态视图可以被捕获，因为免疫荧光分析需要宿主细胞修复离子。然而，免疫荧光成像的一个重要优点是其易于定性和定量分析。这又可用于获得统计学显着的差异，以提供对宿主 - 病原体相互作用的新见解。 荧光图像分析程序是执行3D和4D分析的强大的分析工具。然而，软件及其维护成本高昂使得基于免费开源软件的方法更具吸引力。使用生物分析软件的仔细图像分析是有价值的，因为它证实了视觉分析，并且在分配统计学意义时，增加了给定表型的正确性的置信度。以前，使用免费的ImageJ软件分析SG，这需要手动识别个人SG 4 。在这里，我们提供了一个在细菌SG形成诱导和分析方案使用免费的开源生物图像分析软件ICY（http://icy.bioimageanalysis.org）进行真实感染。生物图像分析软件具有高度适用于SG分析的内置点检测程序。它允许在指定的感兴趣区域（ROI）中对自动检测过程进行微调。这克服了对个别SG的手动分析的需要，并消除了抽样偏差。 许多环境胁迫引起SGs的形成，SG是直径为5 - 6μm的相间致密的胞质结构，非膜结构为0.2-5μm。这种细胞反应在酵母，植物和哺乳动物中进化上保守，并且当全球蛋白质翻译被抑制时发生。它涉及停滞的翻译起始复合物聚集到SG中，其被认为是用于翻译失活的mRNA的保留位置，允许细胞mRNA的子集的选择性翻译。消除压力后，SGs溶解，蛋白质合成的全球速率恢复。 SG由翻译延伸起始因子，涉及RNA代谢的蛋白质，RNA结合蛋白质以及支架蛋白质和参与宿主细胞信号传导的因子2组成 ，尽管精确的组成可以根据施加的应力而变化。诱导SG形成的环境因素包括氨基酸饥饿，紫外线照射，热休克，渗透压休克，内质网应激，缺氧和病毒感染2,7,8 。在理解病毒如何诱导和颠覆SG形成方面取得了许多进展，而关于其他病原体（如细菌，真菌或原生动物病原体）如何影响细胞应激反应1,7尚不清楚。 师哥lla flexneri是人类的革兰氏阴性兼性细胞溶质病原体和严重腹泻或志贺氏菌病的致病因子。志贺氏菌病是一个主要的公共卫生负担，每年导致5岁至9岁的儿童死亡28 000人。 S. flexneri感染结肠上皮，并通过劫持宿主的细胞骨架成分11,12扩散细胞对细胞。上皮细胞的感染支持了胞内溶胶酶的复制，但被感染的巨噬细胞通过炎性细胞死亡过程称为焦灼死亡。感染导致大量招募中性粒细胞和严重的炎症，伴随着热，氧化应激和组织破坏。因此，虽然被感染的细胞受到感染引起的内部应激，例如高尔基体破坏，遗传毒性应激和细胞骨架重排感染细胞也由于炎症过程而受到环境压力。 使用许多SG标记物表征S. flexneri感染对细胞对环境胁迫反应能力的影响已经证明，感染导致SG组成3的定性和定量差异。然而，其他细菌病原体知之甚少。在这里，我们描述了一种感染宿主细胞与细胞溶质病原体S. flexneri ，不同环境胁迫的细胞的应激，SG组分的标记，以及感染SG的形成和组成的定性和定量分析的方法和非感染细胞。该方法广泛适用于其他细菌病原体。此外，SG形成的图像分析可以用于病毒或其他病原体的感染。可用于分析SG感染后的形成或感染对SG形成的影响以响应外源性应激。

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			<td height="21" style="height:21px;width:232px;">Primary Antibodies</td>
			<td style="width:211px;"></td>
			<td style="width:76px;"></td>
			<td style="width:672px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (N20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16377</td>
			<td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (A20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16378</td>
			<td>Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3A (D51F4), origin rabbit (MC: monoclonal)</td>
			<td>Cell Signaling</td>
			<td>3411</td>
			<td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1 in 800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4AI, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-14211</td>
			<td>Recommended by (Ref # 13). Dilution 1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Abcam</td>
			<td>ab186856</td>
			<td>Good stress granule marker in our hands. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Cell Signaling</td>
			<td>3592</td>
			<td>Recommended by Ref # 13. Dilution 1 in 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4G, origin rabbit</td>
			<td>Santa Cruz</td>
			<td>sc-11373</td>
			<td>Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G3BP1, origin rabbit (MC: monoclonal)</td>
			<td>BD Biosciences</td>
			<td>611126</td>
			<td>Widely used SG marker. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tia-1, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-1751</td>
			<td>Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Alexa-conjugated Secondary Antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-goat , origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11055</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-goat, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11057</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21202</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10037</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A31571</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21206</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10042</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shigella flexneri</td>
			<td></td>
			<td></td>
			<td>Available from various laboratories by request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone Casein Soya (TCS) broth</td>
			<td>BD Biosciences</td>
			<td>211825</td>
			<td>Standard growth medium for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCS agar</td>
			<td>BD Biosciences</td>
			<td>236950</td>
			<td>Standard growth agar for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Congo red</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>27215.01</td>
			<td>Distrimination tool for Shigell that have lost the virulence plasmid, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly L lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
			<td>Useful to coating bacteria to increase infection, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Life Technologies</td>
			<td>15630-056</td>
			<td>PH buffer useful when cells are incubated at room-temperature, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>31885</td>
			<td>Standard culture medium for HeLa cells, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Biowest</td>
			<td>S1810-100</td>
			<td>5% supplementation used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td>11140</td>
			<td>1/100 dilution used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Reagent diluent, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium arsenite</td>
			<td>Sigma-Aldrich</td>
			<td>S7400</td>
			<td>Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clotrimazole</td>
			<td>Sigma-Aldrich</td>
			<td>C6019</td>
			<td>Potent stress granule inducer (Note:health hazard, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Electron Microscopy Scences</td>
			<td>15714</td>
			<td>4% PFA is used for standard fixation of cells, application - fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application - permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647-phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A22287</td>
			<td>Dilution is at 1/40, best added during 2ary antibody staining, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleid acid stain used to visualize both the host nucleus and bacteria, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>BR701501</td>
			<td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold</td>
			<td>Thermo Fisher</td>
			<td>36930</td>
			<td>Robust mounting medium that works well for most fluorophores , application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mowiol</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well cell culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td>Standard tissue culture plates, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-mm glass coverslips</td>
			<td>NeuVitro</td>
			<td>1001/12</td>
			<td>Cell culture support for immunofluorescent applications, application - cell support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forceps</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and obust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Programs and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Data analysisand graphing program with robust statistical test options, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Confocal microsope, application - image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>Professional image analysis program, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Data analysis and graphing program, application - data analysis</td>
		</tr>
	</tbody>
</table>

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

细胞成分的荧光成像是调查宿主 - 病原体相互作用的有效工具。病原体可以影响感染细胞的许多不同特征，包括细胞器超微结构，细胞骨架网络组织，以及细胞过程如应激颗粒（SG）形成。病原体如何颠覆宿主过程的特征是发病机理领域的重要和组成部分。虽然可变表型可能容易可见，但是由病原体攻击诱导的细胞结构中的定性和定量差异的精确分析对于定义实验和对照样品之间的统计学显着性差异至关重要。 SG形成是进化上保守的应激反应，导致抗病毒反应，长期以来使用病毒感染进行了研究1 。 SG形成也影响信号级联，可能还有其他未知的结果适应2 。对病毒以外的病原体如细菌病原体的这种应激反应的表征目前是一个新兴的研究领域3 。目前，即使在病毒系统中，SG形成的定量和定性分析尚未被常规使用。在这里，我们描述了诱导和表征未感染细胞中SG形成和细胞感染细胞病原体的细胞的简单方法，其影响响应于各种外源胁迫的SG的形成。 SG形成和组成的分析通过使用许多不同的SG标记和ICY的点检测器插件（开源图像分析工具）来实现。

为了解释和说明本手册中描述的方案，我们将感染或不与细胞溶质病原体S. flexneri的 HeLa细胞中克霉唑诱导的SGs的形象进行了表征。程序概述如图1所示 ，包括在刚果红板上分解的毒力和无毒的柔毛菌 ，细菌制备，感染，环境应激的添加，样品固定和染色，样品成像和定量，以及图像分析。可以使用许多不同的应力来诱导SG形成，并且各种SG标记可用于询问。 eIF3b是一个规范的SG标记，也清楚地染色细胞的细胞质隔室，可用于鉴定细胞边缘。 G3BP1是广泛使用的SG标记，其聚集成SG而没有任何背景染色（ 图 2A ，B </strong&gt;＆ D ）。细胞最好用共焦显微镜成像，覆盖整个细胞深度的堆叠被加载到成像分析免费ICY（ 图 2A -C ）上。为了更好地识别感染的细胞并将细胞置于感染或非感染组中进行后续分析，增加核酸染色强度是有用的（ 图 2D ）。在成像分析软件中，点检测器然后用于识别每个指定的ROI内的SG（ 图 2E ），并且生成显示所有识别的SG的二进制图像（ 图 2F）。 SG分析需要针对分析的每个SG标记进行定制，并且必须仔细选择适当的分析设置。专注于SG marker G3BP1，改变检测点的尺寸要求或改变检测参数的灵敏度将导致不同的结果， 如图3A -C所示 。尺度选择（1 - 3，虽然可以添加尺度）基于像素大小，因此在较高的刻度上，较小的SG不会被计数，而SG的数量将会减少（ 图3 C ）。灵敏度测量范围为1 - 100，最敏感度为100。通过增加灵敏度，检测到的SG数量增加（ 图3C ）。因此，必须找到正确的设置，以尽量减少假阳性和假阴性。最好通过仔细分析每个设置获得的视觉效果。对于G3BP1，灵敏度为100（2 - 100，以红色突出显示）的2的比例为最佳结果。灵敏度较低（50或25）的刻度为2，刻度较小SG不计数（见橙色箭头）。同样地，3的小尺度的小SG不明，而1级的过度采样。重要的是，如果这是有必要的，可以添加额外的量表来集中于大型SG。对于eIF3b，灵敏度为55（2-55）的2的等级为eIF3b（以红色突出显示）提供了最佳效果，尽管红色箭头分别以2 - 50和2 - 55级标高或过采样。 一旦SG分析的参数被正确设置，数据可以以许多方式进行分析（ 图4 ）。斑点检测器给出了在每个ROI内检测到的SG的数量，使得可以比较未感染和感染的细胞（ 图4A ）。类似地，给出了在这种情况下的像素数量的大小，可以从中计算表面积（ 图 4B ）。频率分布它也有助于突显不同细胞群体中SGs大小的变化。在这里， S. flexneri感染细胞中完全不存在大的SG，以及分布中的明确变化变得更清楚（ 图 4C ）。此外，强度（此处显示为最小和最大强度）提供有关分析的SG的质量的信息。对于S. flexneri感染的细胞，SG显着较不强烈。这些分析提供了统计学上相关的信息，当细胞感染有或没有S. flexneri时，响应外源性应激形成的SG的定性和定量性质。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55536/55536fig1.jpg" /> 图1 :用S.Flexneri感染细胞的实验程序的概述分析感染SG形成的影响。在胰蛋白酶大豆肉汤中挑选出刚果红色菌落和非毒性的刚果红色阴性菌落，生长O / N。将过夜培养物传代培养至晚期指数期，然后用PLL涂覆细菌以促进依从性。在12孔板中玻璃盖玻片上生长的宿主细胞用细菌感染30分钟。对照细胞未被感染。将细胞洗涤并用应激诱导剂处理以通过用含应力的培养基替换培养基或使细胞经受诸如加热的胁迫条件来诱导SG形成。然后将细胞固定，透化，用免疫荧光SG特异性标记染色，并使用共聚焦显微镜成像。使用ImageJ进行Z投影，并使用图像分析软件的斑点检测器进行分析。然后分析数据。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55536/55536fig1large.jpg" target="_blank">请点击这里查看大图</a>呃这个数字版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55536/55536fig2.jpg" /> 图2 :在加入克霉唑1小时之前1.5小时感染S.Flexneri的HeLa细胞的点检测分析。 A。 Z投影的免疫荧光图像显示用SG标记eIF3b和G3BP1，DAPI和肌动蛋白染色的细胞。 B。与（A）相同的图像，但没有肌动蛋白染色突出显示使用eIF3b来描绘细胞边界。 C。用斑点检测器模块对图像分析软件进行屏幕截图。 D。感染和未感染细胞的门控，如使用不同渠道所见。看到所有的细菌，强度增加。细胞中存在多于1种细菌的细胞用红色星标记。 E。输出点检测分析个人投资回报率，指示ROI名称，点数及其位置。 F。在ROI中检测到的斑点的图像。刻度棒=30μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55536/55536fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55536/55536fig3v2.jpg" /> 图3 :用于通过不同SG标记检测SG的点检测器的不同尺度和灵敏度设置的比较。 A。放大到感染或不与S. flexneri感染的HeLa细胞，并用DAPI和SG标记G3BP1染色，并以不同的尺度（像素大小截止，1-3）和灵敏度（1-100）进行分析。在不同尺度下分析的未感染和感染细胞中SG数量的图形表示（ <strong<html> &gt; B）和敏感度（ C ）。当改变灵敏度极限而不改变光斑尺寸截止值时，用于相同图像的SG标记eIF3b的SG号检测的视觉（ D ）和图形表示（ E ）。红色写作和红色符号表示最佳参数。红色箭头指向假阳性和橙色箭头，缺乏SG检测。值包括具有标准偏差的平均值。最好的结果以红色突出显示。使用左侧的Wilcoxon秩和检验p值和右侧的方差F检验来选择统计分析。 * = &lt;0.05，** = &lt;0.01，*** = &lt;0.001，ns =无显着性。刻度棒=10μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55536/55536fig3v2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> es / ftp_upload / 55536 / 55536fig4.jpg"/&gt; 图4 :从感染S. Flexneri的克霉唑治疗的HeLa细胞获得的点检测器产生的SG数据的分析。 A。感染和未感染的HeLa细胞中每个细胞的G3BP1 SG的数量。 B。感染和未感染细胞中G3BP1-SG的表面积及其分布频率（ C ）。 D。 G3BP1-SG的最小和最大强度值（任意）。值包括具有标准误差的平均值。使用左侧的Wilcoxon秩和检验p值和右侧的方差F检验来选择统计分析。 * = &lt;0.05，** = &lt;0.01，*** = &lt;0.001，ns =无显着性。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55536/55536fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

我们描述了在用细菌和许多不同的应力攻击细胞后，哺乳动物细胞中应激颗粒形成的定性和定量分析的方法。该方案可用于研究广泛的宿主细菌相互作用中的细胞应激颗粒反应。

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

immunofluorescence-analysis-stress-granule-formation-after-bacterial

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  Institut Pasteur. 我们描述了在用细菌和许多不同的应力攻击细胞后，哺乳动物细胞中应激颗粒形成的定性和定量分析的方法。该方案可用于研究广泛的宿主细菌相互作用中的细胞应激颗粒反应。 

视频: Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

科研

免疫与感染

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

在细菌粘附的研究剪应力

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

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 ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

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