Name: immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells
Uploaded: 2017-07-03T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

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?...

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。

<table><tbody><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>eIF3b (N20), origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-16377</td><td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1:300</td></tr><tr><td>eIF3b (A20), origin goat&amp;nbsp;</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 1:300</td></tr><tr><td>eIF3A (D51F4), origin rabbit (MC: monoclonal)</td><td>Cell Signaling</td><td>3411</td><td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1:800</td></tr><tr><td>eIF4AI, origin goat&amp;nbsp;</td><td>Santa Cruz</td><td>sc-14211</td><td>Recommended by (Ref # 13). Dilution 1:200</td></tr><tr><td>eIF4B, origin rabbit</td><td>Abcam</td><td>ab186856</td><td>Good stress granule marker in our hands. Dilution 1:300</td></tr><tr><td>eIF4B, origin rabbit</td><td>Cell Signaling</td><td>3592</td><td>Recommended by Ref # 13. Dilution 1:100</td></tr><tr><td>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 1:300</td></tr><tr><td>G3BP1, origin rabbit (MC: monoclonal)</td><td>BD Biosciences</td><td>611126</td><td>Widely used SG marker. Dilution 1:300</td></tr><tr><td>Tia-1, origin goat&amp;nbsp;</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 1:300</td></tr><tr><td>&lt;strong&gt;Alexa-conjugated Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A488 anti-goat , origin donkey</td><td>Thermo Fisher</td><td>A-11055</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A568 anti-goat, origin donkey</td><td>Thermo Fisher</td><td>A-11057</td><td>Cross absorbed. Dilution 1:500</td></tr><tr><td>A488 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A-21202</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A10037</td><td>Dilution 1:500</td></tr><tr><td>A647 anti-mouse, origin donkey</td><td>Thermo Fisher</td><td>A31571</td><td>Dilution 1:500</td></tr><tr><td>A488 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A-21206</td><td>Dilution 1:500</td></tr><tr><td>A568 anti-rabbit, origin donkey</td><td>Thermo Fisher</td><td>A10042</td><td>Dilution 1:500</td></tr><tr><td>&lt;strong&gt;Other Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Shigella flexneri&lt;/em&gt;</td><td></td><td></td><td>Available from various laboratories by request</td></tr><tr><td>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><td>TCS agar</td><td>BD Biosciences</td><td>236950</td><td>Standard growth agar for Shigella, application - bacterial growth</td></tr><tr><td>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><td>Poly-L-Lysine (PLL)</td><td>Sigma-Aldrich</td><td>P1274</td><td>Useful to coating bacteria to increase infection, application - infection</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-056</td><td>PH buffer useful when cells are incubated at RT, application - cell culture</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>31885</td><td>Standard culture medium for HeLa cells, application - cell culture</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Biowest</td><td>S1810-100</td><td>5% supplementation used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Life Technologies</td><td>11140</td><td>1:100 dilution used for HeLa cell culture medium, application - cell culture</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td>Reagent diluent, application - cell culture</td></tr><tr><td>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><td>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><td>Paraformaldehyde (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><td>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><td>A647-phalloidin</td><td>Thermo Fisher</td><td>A22287</td><td>Dilution is at 1:40, best added during secondary antibody staining, application - staining</td></tr><tr><td>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><td>Parafilm</td><td>Sigma-Aldrich</td><td>BR701501</td><td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td></tr><tr><td>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><td>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><td>24-well cell culture plate</td><td>Sigma-Aldrich</td><td>CLS3527</td><td>Standard tissue culture plates, application - cell culture</td></tr><tr><td>12 mm&amp;nbsp; glass coverslips</td><td>NeuVitro</td><td>1001/12</td><td>Cell culture support for immunofluorescent applications, application - cell support</td></tr><tr><td>forceps</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><td>&lt;strong&gt;Programs and Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>GraphPad Software</td><td></td><td>Data analysisand graphing program with robust statistical test options, application - data analysis</td></tr><tr><td>Leica SP5</td><td>Leica Microsystems</td><td></td><td>Confocal microsope, application - image acquisition</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>Professional image analysis program, application - data analysis</td></tr><tr><td>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，?...

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

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

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

生物工程

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

利用受刺激的排放损耗 (STED) 显微镜可视化肌动蛋白和微管骨架在 B 细胞免疫突触中的应用

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

子结构分析仪:用于荧光显微镜图像中细胞体快速探索和精确分析的用户友好型工作流程

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

压力处理下细菌生长的多尺度分析

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

生物学

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Source: Aulas, A. et al., <a href="https://app.jove.com/v/55656">Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

一种用于表征和定量哺乳动物应激颗粒的免疫荧光测定

1. Cell Preparation

	Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in&#160;Figure 1A; this can be done using a sterile Pasteur ...