我们感谢Ivanov和Anderson实验室的成员对这份手稿的有益的讨论和反馈。这项工作得到了国家卫生研究院（GM111700和CA168872至PA，NS094918至PI），波兰国家科学中心[授予UMO-2012/06 / M / NZ3 / 00054至WS]的支持。 WS还承认波兰的科学和高等教育部（Mobility Plus计划）和波兰美国富布莱特委员会在美国的研究经费支持。

细胞准备<ol><li>向24孔板的12个孔中加入高压灭菌的盖玻片， 如图1A所示 ;这可以使用连接到真空的无菌巴斯德吸管来接收每个盖玻片。轻轻点击井的盖玻片释放吸力，允许盖玻片落入井中。 </li><li>每孔1×10 5 U-2 OS（U2OS）骨肉瘤细胞，最终体积为500μL培养基。将板一侧移动到一边上下几次，以确保细胞均匀分布。 </li><li>用清洁的P1000小头轻轻按下盖玻片，以确保盖玻片位于井底部，盖玻片下方没有气泡。 </li></ol> 2.压力细胞<ol><li>第二天，目视检查板，以确保细胞均匀分散并均匀地汇合在盖玻片上，作为sa麻烦可能会对结果的重现性产生不利影响。在倒置的组织培养显微镜上使用10X物镜。 </li><li>将培养基预热至37°C。避免向细胞施加冷或热介质，因为这些会导致冷冲击或热冲击。 </li><li> 在预热的介质中稀释药物。对于每种不同的药物浓度，制备含有500μL培养基的2孔。准备另外500μL以允许在移液时丢失。等分4管，每个含有1.5 mL。 <ol><li>对于亚砷酸钠:向含有500μL的每个孔中加入1.5μL的100mM亚砷酸钠（终浓度:100μM）或加入0.75μL的100mM亚砷酸钠（终浓度:50μM）。 </li><li>对于长春瑞滨:向每个含有500μL的孔中加入22.5μL的10mM长春瑞滨（终浓度:150μM）或加入18.75μL的10mM长春瑞滨（终浓度:100μM）。 注意:亚砷酸钠<html>是一种良好表征和常用的应激颗粒诱导剂，因此经典地用作阳性对照。 </li></ol></li><li> 从B1 - 4和C1 - 4井中取出并丢弃培养基。用药物加入培养基并等待60分钟（图1A）。 <ol><li>向孔B1和B2中加入500μL含有100μM亚砷酸钠的培养基。向孔B3和B4中加入500μL含有50μM亚砷酸钠的培养基。 </li><li>向孔C1和C2中加入500μL具有150μM长春瑞滨的培养基。向孔C3和C4中加入500μL含有125μM长春瑞滨培养基的培养基。 </li></ol></li><li>在固定前30分钟，用2μL的嘌呤霉素（1.25mg / mL储备）用5μL的放线菌酮（1mg / mL原液）和第4列的孔处理第2列中的孔。在将板放回37℃的培养箱中之前，将板轻轻摇匀。 </li></ol>细胞固定和免疫荧光注意事项:在实验前，确保甲醇冷却至-20°C，制备缓冲液，包括磷酸盐缓冲盐水（PBS），PBS中5％正常血清（NHS）和4％多聚甲醛（PFA） ）在PBS中。 <ol><li>丢弃介质并用PBS清洗含有盖玻片的孔。根据所使用的药物，妥善丢弃含有药物的培养基可能很重要。请联系当地的环境安全办公室了解具体说明。为了有效地洗涤，用PBS填充挤压瓶，切开尖端，以便轻轻地流动而不是流动紧密，并且在吸取原始的PBS后，使用它将PBS添加到每个孔中。 </li><li>使用〜250μL的4％PFA在室温下轻轻搅动固定细胞15分钟。用PFA完全覆盖盖玻片的顶部; 250μL应该是足够的，但如果没有，添加更多。总是避免直接移到盖玻片上，因为一些细胞（或细胞的压力处理）可以改变附着力，而t他强力的移液可以驱除细胞。 </li><li>取出PFA并正确丢弃。有关如何正确丢弃多聚甲醛的详细信息，请联系当地的环境安全办公室。 </li><li>加入〜250μL甲醇（-20°C），并在室温下轻柔摆动孵育5 min;这两者都渗透和平坦化细胞。 </li><li>通过在4°C的RT O / N处理约250μL的5％NHS 1小时，去除并丢弃甲醇并阻断细胞。有关如何正确丢弃甲醇的详细信息，请联系当地的环境保护局。 </li><li> 对于一级抗体，在5％NHS中稀释抗体。 注意:使用多个SG标记是确认观察到的颗粒是否真正的 SG的关键。可以使用的SG标记的数量取决于显微镜中可用的过滤器。如果标准的绿色，红色和远红色滤光片可用，请以1/250稀释度使用G3BP1，eIF4G和eIF3b。 <ol><li）对于该实验（每个250μL的12个盖玻片 - 总计:3mL的5％NHS），加入以下各物质的12μL:抗G3BP1，抗eIF3b和抗eIF4G。使用温和搅拌孵育一次抗体至少1小时在室温或O / N在4℃。 </li></ol></li><li>用PBS清洗孔3次，并孵育每次洗涤5分钟。 </li><li> 对于二次抗体，在5％NHS中稀释所有二级抗体（1/250稀释）和Hoechst染料（1 / 1,000稀释）。 <ol><li>对于该实验（每个250μL的12个盖玻片 - 总计:3mL的5％NHS），加入以下各物质的12μL:Cy2-小鼠，Cy3-兔和Cy5-山羊（每个的1/250稀释度）并加入3μLHoechst染料。 </li><li>在室温下将盖玻片与第二抗体孵育1小时。在此步骤和以下步骤中，通过用盒子覆盖板或通过将箔放置在组织培养皿的顶部来保护样品免受光照。 </li></ol></li><li>洗我们用PBS洗3次，每次5分钟。 </li><li>使用安装介质将盖玻片安装到标有标签的玻璃载玻片上。将安装介质加热到37°C的热块中10分钟;这降低了粘度，并且更容易移液。使用切割的P200尖端，将25μL每个盖玻片的安装介质移至玻片上;假设显微镜平台允许4-8个盖玻片可以装在一张玻璃片上。使用细镊子，将盖玻片从井中转移到滑块，确保将电池侧面朝下放在安装介质上。使用干净的P200提示，向下按盖玻片。 </li><li>一旦所有的盖玻片都已经安装，使用折叠的实验室组织来清洁多余的安装介质，将实验室组织非常牢固地压在载玻片上。使用含有H 2 O的挤压瓶冲洗多余的安装介质，然后用​​折叠的实验室组织重复印迹以除去水分。 </li></ol> 4. 原位荧光杂交（FISH） <ol><li>使用步骤1和2.1-4作为指导板并处理细胞;仅需要在第1列中压制细胞，因为在以下实验中只需要3个盖玻片。 </li><li>确保制备所有缓冲液（ 即 PBS中4％PFA，70％乙醇水溶液，2x盐水 - 柠檬酸钠（SSC）和5％NHS），将甲醇冷却至-20℃，将杂交烘箱加热至合适的温度。 </li><li>执行步骤3.1-3.3洗涤和固定细胞。 </li><li>通过加入-20°C甲醇使细胞渗透10分钟。 </li><li>取出甲醇，并将盖玻片在70％乙醇中孵育。将板放置在4℃过夜。使用石蜡膜密封板，以防止蒸发。 </li><li>第二天，加入500μL2x SSC，去除乙醇并再次使细胞再水化。在室温下轻轻摇动孵育盖玻片5分钟。 </li><li>取出2x SSC并加500μL2X SSC。温和搅拌下孵育5分钟。 </li><li>将一片石蜡膜平放在杂交烤箱的底部。将25μL杂交缓冲液移至每片盖玻片上的石蜡膜上。从24孔培养皿中取出盖玻片（此时，盖玻片是细胞侧向上），并将盖玻片细胞侧放在杂交缓冲液上。在42℃或65℃下进行15分钟。 注意:在65°C完成此步骤会使细胞RNAs变性;如果polyA被与蛋白质的相互作用掩蔽，则可以改善信号。 </li><li>在杂交缓冲液中稀释生物素 - 寡核苷酸（dT）探针（100ng /μL），浓度为2ng /μL;每盖玻片25μL就足够了。 </li><li>对于每个盖玻片，将25μL含有生物素 - 寡糖（dT）的杂交缓冲液移液到石蜡膜上。用镊子拿起盖玻片，轻轻地将盖玻片的边缘接触到实验室组织上以除去多余的物质杂交缓冲液。将生物素 - 寡糖（dT）将盖玻片转移到杂交缓冲液中;记住要保持单元格面朝下。 </li><li>将盖玻片放在杂交盒中以限制蒸发。在42℃下，将杂交缓冲液中的生物素 - 寡糖（dT）盖玻片孵育60分钟。 </li><li>将2x SSC置于杂交烘箱中，使其平衡至42℃。 </li><li>向24孔培养皿中的孔中加入约500μL加热的2x SSC，并使用镊子将盖玻片转移到孔中。孵育10分钟</li><li>在42℃下重复上述洗涤步骤两次，然后在室温下重复两次。 </li><li>完成步骤3.5-3.10，确保使用链霉抗生物素蛋白荧光染料检测生物素 - 寡糖（dT）而不是一种常规抗体。 </li></ol>用于评估转化状态的雷帕霉素测定<ol><li>板盖U2OS细胞在盖玻片上，如步骤1所示，但每个条件只能盖上一片盖玻片情况，3个盖玻片:未处理的，100μM亚砷酸钠和150μM长春瑞滨）（ 图 1A）。 </li><li>使用步骤2中的协议应激细胞，完成步骤2.1-2.4。 </li><li>固定前5 min，加入2μL嘌呤霉素（库存:1.25 mg / mL;稀释2μL，500μL，最终嘌呤霉素浓度为5μg/ mL）和2.9μL的埃米汀（库存:20μg/ mL;加入将2.9μL与已经含有嘌呤霉素的相同溶液）加入到含有0.5mL的每个孔中。 </li><li>完成上述步骤3.1-3.10，使用抗嘌呤霉素抗体（1/1000稀释）检测嘌呤霉素。理想情况下，在其余通道中使用另外两个SG标记。 </li><li>当量化嘌呤霉素信号时，使用相同的曝光拍摄所有图像。通过使用最亮（不受压力）的样品并使图像尽可能亮而不饱和，选择此曝光。 注意:抗嘌呤霉素荧光信号的强度表示随着嘌呤霉素替代氨基酸，正在进行翻译，并入新生蛋白质，并且强制蛋白质的过早终止。 </li></ol>图像采集与分析<ol><li> 量应力颗粒 <ol><li>为了量化SG，使用40X物镜获取总共&gt; 100个细胞的样品的3 - 5个图像。或者，使用其他目标获取图像，但确保每个盖玻片计数&gt; 100个细胞，并且放大倍数足够大以清楚地显现颗粒。通过将盖玻片分成五个大致相等的区域并从每个区域随机选择一个场（ 图1B ）来实现。同一领域的图像应包括评估标记共定位的所有通道。 </li><li>获取所有图像后，合并频道。有些相机软件会自动执行此操作，而其他相机则需要手动合并;这可以做到ImageJ打开所有图像，然后选择[Image |颜色|合并频道]。 </li><li>使用Hoechst作为核标记，通过计数细胞核的数量手动计数细胞总数。手动计算每个单元格包含多于两个SG的单元格数。 注意:例如，使用合并的三通道图像显示G3BP1（绿色），eIF4G（红色）和Hoechst（蓝色），使用蓝色通道计数细胞核（或细胞数）。然后，将SG阳性细胞计数为每个细胞具有两个或更多个黄色焦点的细胞。颗粒将呈现黄色，绿色为G3BP1，红色为eIF4G，如果共定位，则呈黄色。 </li><li>通过组合来自3-5个独立区域的数据确定SG阳性细胞的百分比，然后计算: SG阳性数/核数）* 100 = SG阳性细胞的百分比</li></ol></li><li> 通过线扫描分析评估共定位 - 手动方法 <ol><li>打开ImageJ的。从（）下载它</li><li>打开ROI管理器:[Analyze |工具|投资回报率经理...] </li><li>在ImageJ中打开合并的图像:[File |打开]。 </li><li>使用位于ImageJ工具栏中的线工具，添加一条通过SG的线，确保在SG之前和之后包含。通过点击ROI管理器窗口中的[添加]将此行添加到投资回报率管理器。 </li><li>将合并的图像分割为不同的通道，以评估每个标记物对颗粒的定位:[Image |颜色|分裂通道];这将合并的图像分割成三个黑白图像。 </li><li>在黑白图像中，确保选中该行;如果图像中出现该行，该行将被选中。如果没有，在ROI管理器窗口中，单击面板中的行;这将突出显示投资回报率管理器窗口中的蓝色线，并使线条在黑白图像上可见。如果行仍然没有出现，请确保"显示所有"是ch进入ROI管理器，然后点击图像中的行。 </li><li>分析线的强度:[Analyze |剧情简介];这将显示"绘图配置文件"窗口，其中绘制了信号的强度。 </li><li>在"绘图配置文件"窗口中，单击[列表]，该列表将显示包含图形中原始数据的窗口。复制此窗口中的所有数据并将其粘贴到电子表格文件中。为此，请选择窗口中的所有数据:[编辑|全选]。复制日期:[编辑|复制]。打开电子表格文件并粘贴数据:[编辑|糊]。 </li><li>分别完成每个通道的步骤6.2.6 - 6.2.8。请确保跟踪正在评估的频道。 </li><li>在电子表格中，生成结果的线形图。按[控制]并点击每列顶部的字母，选择包含数据的列。然后，选择线图:[插入|线形图]。 </li><li>重复此分析多次独立实验中的微量压力颗粒。 </li></ol></li><li> 通过线扫描分析评估共定位 - ImageJ插件 <ol><li>从ImageJ网站下载并安装RGB配置文件工具（https://imagej.nih.gov/ij/macros/tools/RGBProfilesTool.txt）;当正确安装时，工具栏中应显示红色，绿色和蓝色（RGB）的图标。 </li><li>在ImageJ中打开图像（ 例如， Tiff，JPG）:[File |打开]。 </li><li>单击位于ImageJ工具栏中的RGB图标。 </li><li>单击并拖动以添加延伸穿过SG的行，确保包含相邻区域;直方图的图像将显示在弹出窗口中。 </li><li>将数据保存为图像:[File |另存为| TIFF]。或者，导出，然后使用上面的步骤6.2.8从另一个图形程序中绘制数据。 </li></ol></li></ol>

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作者没有什么可以披露的。

如通过多种疾病的免疫组织学研究证明，慢性应激导致不同细胞内病灶的形成。例如，大多数神经变性疾病的特征在于不溶性蛋白质的细胞内聚集体。 SG相关蛋白在这种聚集体中的存在通常是得出这样的焦点是SG的基础。在用新的应激刺激处理的细胞中观察到SG标记阳性细胞质病灶时也得出了类似的结论。该协议提供了一个简单的工作流程来识别真正的 SG。 推定的SGs必须符合若干标准，包括组成和动态特征。首先，SG是包含翻译停滞的mRNP的细胞质病灶。因此，第一种方法是使用两种基于显微技术的方法来表征其组成，即polyA-FISH和IF。 FISH是可视化的可靠方法确定与SG共定位的mRNA。使用寡聚（dT） 40探针来检测在压力条件下包装成SG的聚腺苷酸化mRNA。由于寡核苷酸（dT）不会检测到作为加工体（PB）的核心组分的死亡基因mRNA，因此在细胞质病灶中存在强的寡核苷酸（dT）信号是这些灶是第一个指示（尽管不足以定义）的SG。第二个测试是证明poly（A）信号与其他SG标记（ 例如， G3BP1）的共定位（ 图2A ）。这是通过对SG标记蛋白进行IF来完成的。常规使用SG标记eIF3b，eIF4G和G3BP1，因为eIF3b和eIF4G是已知是48S *复合物的组分的翻译起始因子，并且G3BP1是SG形成所必需的（ 图2B ）。重要的是要强调，需要多个SG标记，因为不同的压力将不同的SG相关因子引入到SG中，并且一些应力可以c可能被误认为SG形成的蛋白质聚集。 第二，SG由于翻译抑制而形成。虽然可以通过不同的方法（ 例如，通过传统的[ 35 S] Met追踪标记）来检测翻译的抑制），但是优选地，因为它检测处理的细胞中正在进行的蛋白质翻译。该技术也与SG标记的标准免疫荧光相容，从而允许同时监测SGs和个体细胞中的翻译抑制程度。 如图3所示 ，SA和VRB都促进SG形成的程度不同。虽然SA在近100％的细胞中引起SG，但是VRB在约75％的细胞中促进SG形成。促进SGs的能力与翻译抑制程度相关:虽然未处理的细胞具有较高的翻译基础水平，但SA完全抑制翻译，但VRB仅为pa中止翻译（ 图3 ）。 第三，SGs是动态的，并且与翻译多核糖体处于平衡状态。在应激前或与应激同时用放线菌酮处理细胞可防止多聚体分解和SG形成。 SG-多聚体平衡是双向的，因为表现出SG的受压细胞可以用放线菌酮处理，以便在有效启动时通过捕获多核糖体或单体中的mRNA来强制进行SG拆卸。一个重要的控制是使用嘌呤霉素，其通过促进过早终止来抑制延长，从而增加可用于SG形成的非多核糖体mRNP的量。当SA或VRB治疗与CHX或puro治疗联合时，观察到SG的剧烈变化（ 图4 ）。因此，评估推定的SG是否溶解于放线菌酮治疗，但增加或不受嘌呤霉素影响是SG鉴定中使用的关键实验参数阳离子。在非常小的，非粘附的原代人骨髓CD34 +细胞中，通过在细胞分离之前处理悬浮液中的细胞，成功证明了EMetine强制的SG拆卸32 。 亚砷酸钠广泛用于通过诱导eIF2-α磷酸化33来触发SG，但是应该仔细滴定 ，因为它靶向许多蛋白质并激活许多信号通路34,35 。不同的细胞系对亚砷酸钠表现出不同程度的敏感性。 U2OS相对敏感（100μM），而COS7，MEFs和HeLa需要200μM。 DU-145 6 ，原发性正常人骨髓CD34 +细胞32 ，人淋巴母细胞36 ，小鼠皮层神经元37和Huh7细胞31需要500-1,000μM。 <p class ="jove_content"&gt;由于SG的分类主要是基于显微镜的方法，因此获取无偏差的质量图像很重要。重要的是以不偏不倚的方式拍摄图像，因为选择具有高或低数量的SG的区域可能会使研究结果偏斜。其他考虑因素包括使用相同曝光拍摄每个通道中的图像;这将确保盖玻片上的强度变化表示该标记的变化。此外，以非饱和度的强度拍摄所有图像，以便可以在整个实验中评估强度。使用相同的显微镜/相机在同一天获取所有图像，因为灯泡强度的变化可以随时间变化和显微镜之间变化。最后，重要的是检查将用于成像幻灯片的显微镜的过滤器和设置。该协议设置为具有过滤器组的显微镜，可实现UV，绿色，红色和远红色通道的可视化。调整可能对于不同的显微镜是必需的。 这里描述的实验工作允许在不同的电池和不同的应力下检测SG。这个工作流程的主要优点是它简单，允许对真正的 SGs进行明确的检测，以及在应激处理的细胞中同时探测细胞翻译。该协议使用U2OS细胞，但可以修改不同的细胞系或甚至原代细胞。关于应力颗粒研究的一个最新的综合细胞列表是11 。在药物治疗当天应调整细胞数达到80％的汇合度。药物治疗的浓度和持续时间也应调整，因为细胞反应不同。另外，虽然这个协议设置为24孔格式，它可以调整到任何尺寸的板或盘，任何大小的盖玻片可以平躺在电镀和药物治疗NT。稳定表达SG / PB标记的细胞系已经用于高通量，基于siRNA的屏幕33和活细胞成像38中 。使用用于内源性SG标记PABP染色的HeLa细胞来检测基于图像的筛选以检测化学化合物阻断SG组件39 。预计此工作流将在以后的研究和应用中有用。

细胞采用许多机制来应对压力。一些反应发生在转录后水平，并涉及调节mRNA翻译和/或稳定性1,2 。应激调节的mRNA翻译阻滞和降解与特异性非膜细胞病灶的形成相关，其最好的特征是应激颗粒（SGs​​） 3 。 SG是细胞质焦点，其将非翻译的mRNP浓缩在对胁迫（ 例如，氧化，热休克，营养物饥饿和病毒感染）反应的缓冲细胞中。除了非翻译mRNA之外，SG还含有翻译起始因子，RNA结合蛋白和各种信号转导蛋白。 SG是抑制蛋白质翻译和RNA代谢改变的生物标志物，并与细胞存活和细胞凋亡信号通路有关和核工程5 。 SG是动态实体，它们的形成与细胞翻译的状态紧密相关6 。尽管它们看起来很稳固，但大多数SG蛋白质组件都能快速穿梭进出，停留时间为秒。虽然SG持续了几分钟到几个小时，但其大多数组件都处于快速通量。翻译起始的抑制和随后的翻译多核糖体的拆卸促进了SG的形成;因此，SGs与翻译多核糖体处于平衡状态。这种多聚体/ SG平衡是将真正的 SG与其他应激诱发的病灶区分开来的关键6,7 。 抑制翻译起始需要转录含有mRNA，翻译起始因子（eIF）和40S核糖体亚基（s）的翻译能力的起始前复合物<html>称为48S复合物）转化为可以聚结成SG 4,6的翻译失活复合物（所谓的48S *复合物）。 SGs通过在48S复合物形成上游的两个不同步骤的翻译阻滞来促进:干扰cap结合eIF4F复合物的功能（ 例如靶向其eIF4A亚基）或翻译起始因子eIF2的α亚基的磷酸化，由一个介导的或更多的四种eIF2激酶。停滞的48S复合物（ 即 40S核糖体亚基和选择的翻译起始因子）的存在是SG8,9的标志。 SG已经与各种病理状态相关联，例如病毒感染，神经变性，自身免疫和癌症3,10,11 ，ss ="xref"&gt; 12，13。几种SG成分的突变形式与神经变性疾病（ 例如，肌萎缩性侧索硬化症）相关，其中神经元显示可能在神经元死亡中起主要作用的细胞内病理性包涵体13 。一些病毒劫持SG成分以抑制SG形成和增强病毒复制14 。最近的研究还将SG与癌症15联系起来，并将化疗和放疗治疗的癌细胞存活10,16,17,18,19,20。虽然这些发现激发了对SG生物学的极大兴趣，但许多已发表的报告缺乏重要的控制来区分真正的 SG的形成和其他压力引起的病灶。 SG最初描述为由选定的mRNA结合蛋白（RBP）组成的非膜性细胞质病灶，包括T细胞细胞抗原1（TIA-1）和Poly-A结合蛋白（PABP）;翻译起始因素;聚腺苷酸化mRNA;和小核糖体亚基4,6,21,22。传统上，它们的组成通过诸如免疫染色和荧光标记蛋白的异位表达的技术23,24来确定。由于其高度动态的性质，SG蛋白和/或mRNA的免疫定位仍然是检测SG 23,24的定义方法。迄今为止，许多RBP和其他蛋白质（超过120种不同的蛋白质）被描述为SG组分11 。多少SG-局部蛋白质以应激依赖性和独立性方式改变其定位，并且可以在其他细胞内区室和灶点积累，选择正确的SG标记物并采用功能标准来区分SG与其他类型的应激诱导灶。 真正的 SGs含有mRNA，翻译起始因子和小核糖体亚基，并且与翻译处于动态平衡。 这个简单的工作流程旨在确定应激诱发的病灶是否真正的 SGs。该工作流程包括使用U2OS细胞的几种实验方法，通常用于研究SG的细胞。这些细胞是理想的，因为它们具有大的细胞质，相对平坦，并且牢固地附着在玻璃盖玻片上。其他细胞类型可用于研究SG，但重要的是要注意，时间，药物浓度和SG-成核蛋白丰度的差异可以改变动力学和组合sition。另外，一些细胞通过形成细胞表面泡泡来响应多聚甲醛固定，然后产生引起一些SG标记物的点状定位的皱纹外观;没有仔细分析，这些可以被分类为SG。这突出了必须评估将应激诱发的病灶识别为真正的 SG所需的所有标准。长春瑞滨（VRB）是一种促进SG 20的癌症治疗剂，以及砷酸钠（SA），一种健壮且性能良好的SG诱导剂，被用作本协议实验的应力。 标准SGs包含多个SG标记（蛋白质和mRNA），共定位于细胞质病灶。该方案同时使用免疫荧光和荧光原位杂交（FISH）检测蛋白质标记和聚腺苷酸化mRNA 22的定位。简言之，间接免疫荧光使用抗体（即即一级抗体）对于给定的蛋白质是特异的，以在细胞内检测它。然后，附着于荧光染料的二抗（通常是物种特异性）识别第一抗体并揭示靶蛋白定位。荧光显微镜用于检测细胞内的局部信号。使用不同物种产生的抗体并用不同颜色的二级抗体进行检测，可以检测多种抗体的共定位，表明它们的蛋白质靶标位于同一位置23 。选择已被验证以在真正的 SG上共定位的标记是重要的。 FISH使用与特定RNA或DNA序列碱基对的标记探针25 。为了检测mRNA，该方案使用与mRNA的polyA尾杂交（或碱基对）的生物素化寡核苷酸（dT） 40探针（ 即 polyA FISH）。然后使用荧光偶联的链霉亲和素检测生物素化的探针，因为链霉亲和素对生物素具有高亲和力。当评估SG时，重要的是将polyA FISH与免疫荧光检测SG标记物结合，如真正的 SG，两个信号应共定位。 使用该协议，共定位以多种方式进行评估。在多通道（ 即 RGB:红色，绿色和蓝色）图像中，colocalization将改变重叠信号的颜色（ 例如， colocalized红色和绿色出现黄色） 23 。另外，使用线扫描分析图形化定量共定位，其中在给定线8,20上测量每个颜色的强度。该协议描述了使用ImageJ 26的两行扫描分析程序。一个程序是手动的，经过整个过程，另一个使用宏，或者一个自动执行手动步骤的简单程序。重要的是要通过手动程序来了解宏程序。 SGs形成在翻译被压制的单元格中;因此，与未处理的细胞相比，具有SG的细胞应该显示出降低的全局翻译水平。实验上，使用了尿布溴霉素。在固定之前，将嘌呤霉素和埃马汀加入细胞持续一段时间，使嘌呤霉素掺入活跃形成的多肽中，导致终止27,28 。需要用emetine进行治疗以防止再次起搏29 。然后可以使用抗嘌呤霉素抗体检测嘌呤霉素，给出活性翻译的快照。使用这种方法是因为它很快，不需要用缺乏特定氨基酸的介质（并且潜在地预处理细胞）的预饥饿，并且揭示了蛋白质翻译的亚细胞定位。已经使用其他方法，特别是使用修饰的氨基酸类似物，例如甲硫氨酸类似物L-叠氮基低聚维生素（AHA），与"click-it"化学30相结合 ，表明含有SG的细胞显示比相邻细胞31低得多的翻译。然而，该技术需要甲硫氨酸饥饿，然后脉冲标记15-30分钟。蛋氨酸饥饿构成额外的压力，而长的标签时间（ 即 15-30分钟）导致测量累积而不是正在进行的翻译，并且还允许新合成的蛋白质从其合成位点移动到其最终目的地内细胞。相比之下，番红霉素更快，并且与任何培养基（例如用于通过葡萄糖饥饿诱导SG的无葡萄糖培养基）相容。 规范SGs处于动态平衡状态积极翻译多核糖体。这可以通过用稳定或不稳定多核糖体的药物处理样品来实验地评估，从而在SG和多核糖体之间平衡23 。通过将核糖体"冻结"到mRNA上，环己酰亚胺（或其类似的表现）阻断延伸，从而减少能够形成SG的可用非多核糖体mRNP的库。实验上可以通过以下两种方法使用:在应力之前加入放线菌酮（或埃米汀）以防止SG形成，或者在形成SG之后加入放线菌酮（或埃米汀），即使没有除去应激剂，也会导致SG拆卸停止早产复合物缓慢进入多聚糖部分。相比之下，嘌呤霉素引起过早终止并促进多聚体拆解，增加能够组装成SG的起始mRNA的集合。实验上，嘌呤霉素治疗会增加SG的数量或降低threshold，其响应于分级应激或药物剂量形成。当评估嘌呤霉素的作用时，重要的是使用目的药物的亚最大水平，因为嘌呤霉素预期会增加药物的作用并增加显示SGs的细胞的百分比 - 如果药物不能发生，则不会发生/压力最初导致100％细胞中的SG。这与放线菌酮处理形成对照，当约95％的细胞初始显示SG时，分解SG并且最好地工作，以便可以观察到并且准确地量化最大可能的降低。 该协议提供了研究哺乳动物细胞中SGs的框架。方法包括:（1）免疫荧光染色和ImageJ分析，评估推定的SGs中经典SG相关标记eIF4G，eIF3b和Ras GTPase激活蛋白结合蛋白1（G3BP1）的共定位; （2）寡聚（dT）荧光原位杂交（polyA FISH）检测多聚腺苷酸化mRNA; （3）放线菌酮和嘌呤霉素处理，以确定应激诱导的病灶是否与多核糖体处于动态平衡状态;和（4）番红霉素以评估含有推定的SG的细胞的翻译状态。在一起，这些测定法可以确定应激诱导的病灶是否可归类为真正的 SG。

<table><tbody><tr><td>U-2 OS</td><td>ATCC</td><td>ATCC&amp;reg;HTB-96&amp;trade;</td><td>Commonly written &quot;U2OS&quot;&lt;br/&gt; Culture at 37 &amp;deg;C under 5% CO&lt;sub&gt;2&lt;/sub&gt; in 10% FBS/DMEM</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Corning</td><td>10-013-CV</td><td>Contains 4.5 g/L glucose, pyruvate, and phenol red&lt;br/&gt; Supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin&lt;br/&gt; Prewarm prior drug(s) dilution</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442-500ml</td><td>Used to supplement media</td></tr><tr><td>HEPES (1 M)</td><td>Thermo Fisher Scientific</td><td>15630-080</td><td>Used to supplement media</td></tr><tr><td>penicilline streptomycine</td><td>Sigma</td><td>P0781-100ml</td><td>Used to supplement media</td></tr><tr><td>24-well plate</td><td>Costar</td><td>3524</td><td></td></tr><tr><td>lab tissues (kimwipes)</td><td>KIMTECH SCIENCE</td><td>34120</td><td></td></tr><tr><td>Coverglass for Growth Cover Glasses</td><td>Fisher Scientific</td><td>12-545-82</td><td>Autoclaved before use&lt;br/&gt; Keep sterile in the tissue culture hood</td></tr><tr><td>Sodium (meta)arsenite &amp;ge;90% (SA)</td><td>Sigma</td><td>S7400-100G</td><td>Dissolve in water to 1 M concentration, then dilute to 100 &amp;micro;M as a working stock</td></tr><tr><td>Vinorelbine (VRB)</td><td>TSZ CHEM</td><td>RS055</td><td>Dissolve in water to 10 mM</td></tr><tr><td>Puromycin</td><td>Sigma</td><td>P9620</td><td>Used to assess translation level (ribopuromycylation)&lt;br/&gt; -Used to assess SG connection with active translation&lt;br/&gt; Dilute in water to 10 mg/mL</td></tr><tr><td>Emetine dihydrochloride hydrate</td><td>Sigma</td><td>E2375</td><td>Used in combination with puromycin to assess general translation level&lt;br/&gt; Used to assess SG connection with active translation&lt;br/&gt; -Dilute in water to 10 mg/mL</td></tr><tr><td>Cycloheximide (CHX)</td><td>Sigma</td><td>C4859</td><td>Used to assess SG connection with active translation&lt;br/&gt; Dilute in water to 10 mg/mL</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza / VWR</td><td>95042-486</td><td>Wash buffer for immunofluorescence</td></tr><tr><td>Paraformaldehyde reagent grade, crystalline (PFA)</td><td>Sigma</td><td>P6148-500G</td><td>Make a 4% solution in hot PBS in fume hood, stir until dissolved&lt;br/&gt; Aliquots can be stored at -20 &amp;deg;C for several months&lt;br/&gt; Hazardous, use ventilation&lt;br/&gt; Discard in special waste</td></tr><tr><td>Methanol, ACS</td><td>BDH / VWR</td><td>BDH1135-4LP</td><td>Prechill to -20 &amp;deg;C before use&lt;br/&gt; Discard in special waste</td></tr><tr><td>Normal Horse Serum (NHS)</td><td>Thermo Fisher Scientific</td><td>31874</td><td>Dilute to 5% in PBS&lt;br/&gt; Add sodium azide for storage at 4 &amp;deg;C&lt;br/&gt; Blocking solution for immunofluorescence</td></tr><tr><td>Ethanol, Pure, 200 Proof (100%),USP, KOPTEC</td><td>Decon Labs / VWR</td><td>89125-188</td><td>Dilute to 70% with water</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>mouse anti G3BP1 antibody (TT-Y)</td><td>Santa Cruz</td><td>sc-81940</td><td>Store at 4 &amp;deg;C&lt;br/&gt; 1/100 dilution</td></tr><tr><td>rabbit anti eIF4G antibody (H-300)</td><td>Santa Cruz</td><td>sc-11373</td><td>Store at 4 &amp;deg;C&lt;br/&gt; 1/250 dilution</td></tr><tr><td>goat anti eIF3&amp;eta; (N-20) antibody</td><td>Santa Cruz</td><td>sc-16377</td><td>eIF3&amp;eta; is also known as eIF3b&lt;br/&gt; Store at 4 &amp;deg;C&lt;br/&gt; 1/250 dilution</td></tr><tr><td>mouse anti puromycin 12D10 antibody</td><td>Millipore</td><td>MABE343</td><td>Store at 4 &amp;deg;C&lt;br/&gt; 1/1,000 dilution</td></tr><tr><td>Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson Immunoresearch</td><td>715-225-150</td><td>Reconstitute in water per manufacturer&amp;rsquo;s instructions then store at 4 &amp;deg;C&lt;br/&gt; 1/250 dilution</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson Immunoresearch</td><td>711-165-152</td><td>Reconstitute in water as per manufacturer&amp;rsquo;s instructions then store at 4&amp;deg;C&lt;br/&gt; 1/2,500 dilution</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L)</td><td>Jackson Immunoresearch</td><td>705-175-147</td><td>Reconstitute in water as per manufacturer&amp;rsquo;s instructions then store at 4&amp;deg;C&lt;br/&gt; 1/250 dilution</td></tr><tr><td>Hoechst 33258 solution</td><td>Sigma</td><td>94403-1ML</td><td>Incubate with secondary antibodies&lt;br/&gt; Stock solution 0.5 mg/mL in dH&lt;sub&gt;2&lt;sub&gt;0, protect from light&lt;br/&gt; -Store at 4 &amp;deg;C</td></tr><tr><td>Cy3 Streptavidin</td><td>Jackson Immunoresearch</td><td>016-160-084</td><td>Reconstitute in water per manufacturer&amp;rsquo;s instructions, then store at 4 &amp;deg;C&lt;br/&gt; 1/250 dilution</td></tr><tr><td>oligo-(dT)&lt;sub&gt;40x&lt;/sub&gt; probe biotinilated</td><td>Integrated DNA Technologies (IDT)</td><td>/</td><td>Reconstitute in water to 100 ng/mL, aliquot and store at -20 &amp;deg;C&lt;br/&gt; Dilute in hybridation buffer 1/50 prior to use&lt;br/&gt; Custom order</td></tr><tr><td>hybridation box</td><td>/</td><td>/</td><td>Make a moisture chamber with any storing slide box by adding humidified paper on the bottom&lt;br/&gt; - prewarm the box prior use</td></tr><tr><td>20x SSC buffer</td><td>Thermo fisher Ambion</td><td>AM9763</td><td>Dilute to 2x SSC using RNase Free water (DEPC treated)&lt;br/&gt; Store at RT&lt;br/&gt; Wash buffer for FISH</td></tr><tr><td>PerfectHybPlus Hybridization Buffer</td><td>Sigma</td><td>H7033-50ml</td><td>Block and probe incubation buffer for FISH</td></tr><tr><td>slide mounting media</td><td>home made</td><td>/</td><td>Mix 5 g of &amp;ldquo;cold-soluble&amp;rdquo; poly(vinyl alcohol) in 20 mL of PBS&lt;br/&gt; Mix by sonication, followed by stirring O/N at RT&lt;br/&gt; Add 5 mL of glycerol and 0.2 mL of 20 % sodium azide, and stir for 16 h at RT&lt;br/&gt; Centrifugation at 20,000 g for 20 min, discard large pellet&lt;br/&gt; Aliquot viscous liquid, long-term storage at -20 &amp;deg;C, 1 week at 4 &amp;deg;C</td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>Poly(vinyl alcohol)</td><td>Sigma</td><td>P-8136-250G</td><td>Reagent to make vinol</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-100ML</td><td>Reagent to make vinol</td></tr><tr><td>sodium azide</td><td>Fisher Scientific</td><td>S2002-5G</td><td>Preservative agent for blocking solution and vinol&lt;br/&gt; Make a 20% dilution in water</td></tr></tbody></table>

methods to classify cytoplasmic foci as mammalian stress granules

细胞经常受到突然环境变化的挑战。应激颗粒（SGs​​），在暴露于应激条件的细胞中形成的细胞质核糖核蛋白复合物涉及细胞代谢和存活的各个方面。 SGs调节细胞信号通路，转录后基因表达和应激反应程序。这些含mRNA的颗粒的形成与细胞翻译直接相关。 SG组件由抑制翻译起始触发，SG拆卸通过翻译激活或抑制翻译延伸来促进。 SG组合进一步突出了这种关系。核心SG组分是失速的翻译起始前复合物，mRNA和选定的RNA结合蛋白（RBP）。 SG装配的目的是通过隔离翻译停滞的内部管理mRNA来保存细胞能，从而允许应激反应蛋白的增强翻译。另外对核心成分，如停滞的翻译早产复合物，SG含有大量的其他蛋白质和信号分子。 SG形成的缺陷可以削弱细胞对应激的适应性，从而促进细胞死亡。 SG和类似的含RNA颗粒已经与许多人类疾病相关，包括神经变性疾病和癌症，导致最近对分类和定义RNA颗粒亚型的兴趣。该方案描述了表征和量化哺乳动物SG的测定法。

应激诱发的灶不一定是SG。 SG被分类为含有mRNA，翻译起始因子和RNA结合蛋白的细胞质病灶，并且与主动翻译处于平衡状态。上述方案可用作模板来表征给定的应激是否诱导真正的 SG。 SG与其他已知的SG标记物共定位，包括蛋白质和mRNA两者。 SA和VRB诱导含有G3BP1，eIF4G和eIF3b的细胞质病灶（ 图2A ）。使用线扫描分析和放大倍率增加的单通道图像来确认这些标记的共定位（ 图2A ）。另外，这些病灶含有如PolyA FISH所示的Mrna，寡核苷酸（dT）信号与G3BP1免疫荧光信号共定位（ 图2B ）。显示polyA-FISH是至关重要的信号与已知的SG标记重叠，因为应激可以促进多种类型的病灶。 SG在翻译抑制状态期间发生。 VRB和SA均促进了翻译抑制状态，如用实验性评估的抗尿黄素（ 图3 ）。 真正的 SGs正在动态平衡，主动翻译多核糖体。 SA-和VRB诱导的SGs与CHX一起溶解，CHX将多聚核苷酸捕获在mRNA上，从而停止多聚体拆解和防止SG（ 图4A，4B ）。相反，用puro处理后，诱导更多的SA和VRB SG，因为puro促进多聚体拆解，从而有利于SG形成（ 图4A，4C ）。 总之，与阳性对照SA一样，VRB诱导真正的 SGs:（1）与已知的SG标记共定位，（2）包括蛋白质和mRNA（ Figu<html> （3）在平移压迫的细胞中发现（ 图3 ），和（4）与主动翻译处于动态平衡状态（ 图4A-4C ）。 <img alt="图1" src="/files/ftp_upload/55656/55656fig1.jpg" /> 图1:实验图。 （ A ）如前所述，将种子细胞置于预先置于24孔板中的盖玻片上。用SA或VRB处理A，B行60分钟，使用浓度如μM所示。 60分钟后，向药物培养基中加入CHX（终浓度为20μg/ mL的第2列）或puro（20μg/ mL嘌呤霉素用于第4列）。在固定和染色之前，继续孵育30分钟。 （ B ）盖玻片图，表示应进行成像计算的字段。盖玻片分为五个大致相等的区域;在每个地区拍摄一幅图像。"https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55656/55656fig1large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55656/55656fig2.jpg" /> 图2:含有SG标记anPoly（A）mRNA的SA和VRB诱导细胞质Foci。 （ A ）对G3BP1（绿色），eIF3b（红色）和eIF4G（蓝色）进行免疫染色的U2OS细胞。细胞用100μMSA或150μMVRB处理60分钟或未处理（对照）。缩放区域被放大（2X），并以黑色和白色（下图）显示为单独的通道。该图表示区域（白线）颗粒的线扫描分析，并显示重叠或共定位的程度。比例尺表示10μm。 （ B ）与免疫染色偶联的PolyA-FISH利用寡聚（dT） 40探针（红色）检测polyA mRNA，使用抗G检测G3BP1（绿色）3BP抗体，使用Hoechst染料（蓝色）检测核DNA。缩放区域（2X），单通道图像和线扫描分析显示共定位。刻度棒=10μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55656/55656fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55656/55656fig3.jpg" /> 图3:SA和VRB治疗原因翻译抑制。在指定的治疗后，用嘌呤霉素脉冲标记5分钟的U2OS细胞的免疫荧光。嘌呤霉素（绿色），eIF3b（红色）和用Hoechst染色的核DNA（蓝色）。细胞用100μMSA或150μMVRB处理60分钟或未处理（对照）。刻度棒=10μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55656/55656fig3large.jpg" target="_blank">请点击这里查看更大的经文</a>这个数字的离子。 <img alt="图4" src="/files/ftp_upload/55656/55656fig4.jpg" /> 图4:与多聚体动态平衡的SA和VRB诱导细胞质Foci。 （ A ）描述多核糖体/ SG关系的一般方案。 （ BC ）使用Hoechst（蓝色）染色G3BP1（绿色），eIF3b（红色）和核DNA的U2OS细胞。在加入（ B ）CHX（放线菌酮，20μg/ mL），（ C ）puro（嘌呤霉素，10μg/ mL）之前，用100μMSA或150μMVRB处理细胞60分钟或未处理（对照） ），或没有添加剂另外30分钟的孵育。数字对应于代表性实验中具有SG的细胞的百分比。刻度棒= 25μm，（BC）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55656/55656fig4large.jpg" target="_blank">请点击这里查看大图</a>版本的这个数字。

Watch this Scientific Journal Video about Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules at JoVE.com

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

应激颗粒（SG）是在暴露于各种应激的细胞中形成的非膜细胞质结构。 SGs包含mRNA，RNA结合蛋白，小核糖体亚基，翻译相关因子和各种细胞信号传导蛋白。该协议描述了一种使用几种实验方法来检测，表征和量化真正的 SG的工作流程。

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 ...

methods-to-classify-cytoplasmic-foci-as-mammalian-stress-granules

Universidad San Sebastian

Research

JoVE Journal

Biology

14.5K 次观看.  Brigham and Women's Hospital. 应激颗粒（SG）是在暴露于各种应激的细胞中形成的非膜细胞质结构。 SGs包含mRNA，RNA结合蛋白，小核糖体亚基，翻译相关因子和各种细胞信号传导蛋白。该协议描述了一种使用几种实验方法来检测，表征和量化真正的 SG的工作流程。 

视频: Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

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

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

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 ...

免疫与感染

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

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 ...

生物工程

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

可视化细胞质流动过程中单细胞伤口愈合<em&gt;蓝斑宏声</em

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

翻译起始的分析在应力条件由多聚核糖体剖析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

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 ...

An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Source: Aulas, A. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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 ...

将细胞质Foci分类为哺乳动物应激颗粒的方法

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