Name: live cell imaging and 3d analysis of angiotensin receptor type 1a trafficking in transfected human embryonic kidney cells using confocal microscopy
Uploaded: 2017-03-27T13:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy at JoVE.co

在这个手稿中提出的研究工作得到了补助R01 HL121456-01A1凯瑟琳桑德伯格支持。这项工作除了由徕卡SP8的AOB在乔治敦大学医学中心的显微镜和影像共享资源（MISR），这部分是由来自美利坚合众国国立卫生研究院P30-CA051008资助的可用性得到了支持。我们非常感谢彼得·约翰逊徕卡SP8成像援助，并在乔治敦大学的托马斯Coate生物系实验室使用蔡司LSM880与由卡尔蔡司显微镜LLC阿尔玛·阿诺德提供成像支持。在此稿件内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

第一天 1.细胞接种<ol><li>准备完整的贝科的改良Eagle培养基（DMEM）。添加50毫升胎牛血清，5毫升青霉素/链霉素，5毫升L-谷氨酰胺至450毫升的DMEM，以使补充有胎牛血清（10％）的完全DMEM 500毫升，青霉素/链霉素（1％）和L-谷氨酰胺（1％）。 </li><li>种子人胚肾（HEK）细胞，6-11 50,000 /以及在完全DMEM的8孔腔玻璃罩系统通道数量。 </li><li>保持在37℃的腔幻灯片在具有5％CO 2的24小时的湿润培养箱。 </li></ol>第二天 2 - 脂质体<ol><li>使用无菌条件下与生物安全2级橱柜新鲜使在1A R-GFP质粒和脂质体的解决方案。 </li><li>稀释2μL质粒DNA在900纳克/μL原液在178μL低血清媒体，实现了concentrat大约10纳克/微升质粒DNA的离子。 </li><li>稀释的脂质体原液4.5μl，以175.5微升低血清媒体创造了1:40稀释的股票。 </li><li>添加脂质体溶液的质粒溶液，并通过使用1毫升吸移管向吸取上下20次来混合转染溶液混合。 </li><li>孵育混合物，在室温下（25-27℃）30分钟。 </li><li>而该混合物孵育，用1毫升吸移管除去介质慢慢和慢慢加入1×磷酸盐缓冲盐水（PBS），其预热至37℃的400微升。 </li><li>温和地去除PBS，并用200微升/孔的降低血清培养基预热至37℃更换。 注意:洗涤细胞时，为了避免从盖玻片细胞脱离由于通过移液诱导的剪切应力要小心在所有步骤。 </li><li>保持在37℃下将细胞在具有5％CO 2的30分钟的潮湿培养箱中。 </li><li>一旦孵育期结束后，添加80微升转染混合物的每个孔中，孵育所述细胞为4.0-4.5 h但不超过5在具有5％CO 2的潮湿培养箱中于37℃小时。 </li><li>转染潜伏期结束后，用1毫升吸移管慢慢移除腔室中的介质，并轻轻地用300微升/ 37℃，在湿润的培养箱中5％的CO 2替换以及完全DMEM的孵育细胞24小时。 </li></ol>第三天 3.血清饥饿<ol><li>取出介质轻轻地用1毫升移液器和无血清DMEM的300微升慢慢代替无酚红。 </li><li>保持在37℃的室中，用5％ 的 CO 2的潮湿培养箱中10-12小时。 </li></ol>第四天 4.活细胞成像<ol><li> 染色的细胞内区室（在此情况下，溶酶体）。 <ol><li>阿博UT 30分钟于成像溶酶体中的细胞，从1μM的荧光染料原液添加22.5微升之前（0.5 1微升毫荧光染料在无血清DMEM的499.5微升稀释无酚红）到每个孔中作最后的染料浓度的75纳米。 </li><li>在5％的CO 2的潮湿培养箱中孵育25-30分钟，在37℃，然后轻轻地卸下染料和慢慢无酚红加入新鲜无血清DMEM的300微升。 </li></ol></li><li> 准备用于成像显微镜。 <ol><li>调整阶段，以避免一自动显微镜的启动过程中对透镜的损坏。 </li><li>打开显微镜功率，扫描仪功率，激光功率，然后激光发射，最后的金属卤化物灯为视觉观察（参见共聚焦显微镜的制造商说明书）。 </li><li>打开图象处理软件，并在氩（488纳米）的激光电源，打开软件，翻到30-50％。注意权力取决于激光的年龄。在实验中关键的是要保持恒定的设置。 </li><li>打开红色发射激光器（ 如 568纳米或594纳米）。 </li><li>组影像格式到12位的深度。 </li><li>设置了氩激光线488纳米和568纳米或594纳米的激光1艾里斑单元针孔。如果GFP是非常暗淡，使用2艾里单位设置，但适当匹配的通道。 </li><li>当使用二向色过滤器集，选择GFP和荧光染料适当发射波长。为680nm的红色发光荧光染料 - 或者，在对GFP的499-580纳米和EM 599设置的发射波长（EM）。 </li><li>检查通过流血不发生交叉。关闭每一个激光依次检查每个都发射声道。当氩激光关闭时，绿发射应为零。当绿色发光激光器是关闭的，所述红色发射的信道应该是零。 注:最安全的方法是将图像中的每个单独的轨道（色曲无穷区间）。 </li><li>如果需要，包括明通道。避免差动对比成像（DIC），因为它可以歪斜共定位测量。 </li><li>对于实时成像，选择行顺序，而不是帧顺序。 </li></ol></li><li> 选择单元格进行成像。 <ol><li>利用1.4数值孔径（NA）的目标，诸如63X / 1.4 NA油浸物镜到图像的细胞（或40X / 1.4 NA）。中心的目标和添加高粘度，低自体荧光浸油下降到目标。 </li><li>从培养箱与转染的细胞转移室到显微镜的阶段。小心保持幻灯片平整，确保室很好就位，并限制其运动。 </li><li>移动目标，直到油的降刚好接触滑动的底部;正确的焦平面接近这个位置。在自动显微镜，保存该位置以及降低位置以用于对物成像实验ainder，以方便更换样品。 </li><li>选择暗淡细胞。细胞过度在1A的R-GFP将误导在细胞受体和破坏正常功能。 </li><li>确认该细胞的轮廓，通过质膜中定义，是可见的并且优选不与其它细胞重叠。 </li><li>聚焦和在视场中心的细胞。 </li><li>切换到共聚焦模式。 </li><li>选择快速成像模式。随着对焦点的控制，一方面和其他在舞台上的XY控制，焦点和中心的可视化显示器感兴趣的细胞。 </li><li>确认所关注的细胞通过聚焦上下贯通该单元被很好的普​​及和其底部或腹侧与盖玻片紧密并置。 </li></ol></li><li> 设置的Z轴阶段参数图像在3D的细胞。 <ol><li>选择的Z堆栈的顶部和底部对话框（或同等学历）开按钮，并集中到c的底部埃尔然后按"开始"。然后，集中到细胞，按顶部的"终结"。重新检查整个小区被包括在z栈。 </li><li>在Z-步长为1.0微米。 </li></ol></li><li> 设置成像设置。 <ol><li>选择放大2，这取决于透镜，使最终的像素尺寸为约0.279微米×0.279微米至0.14微米×0.14微米在XY尺寸。 </li><li>对于在该实验HEK细胞，这取决于细胞的亮度扫描速度设定为每像素大约1微秒，然后选择2的平均或2积累由线。 </li><li>调整激光强度，并获得用于成像GFP和荧光染料。在一般情况下，counterstains将变得更亮，因此，光电倍增管检测器（光电倍增管），而不是砷化镓磷化物（的GaAsP）或混合探测器（HYD）应该被用于该第二色彩信道而喘气和路政署探测器是必需的GFP的信道。如果路政署或磷砷化镓检测ORS被用于荧光染料，施加谨慎并在极低功率的激光启动。 </li><li>扫描和扫描的次数的设定时间，例如，每30-60结束了25分钟或更长的时间才能看到溶酶体在1A的R-GFP的定位。 </li><li>设置自动对焦维持一段时间焦平面稳定性。 </li></ol></li><li> 开始成像实验。 <ol><li>在5微克/μL（4.7793毫米）提前100X血管紧张素II准备股票并加入这个2.1μL到998μL介质（10微米）。 </li><li>通过立即成像（终浓度为100nM）之前加入配体（血管紧张素II）的100倍原液的3微升至孔含有300微升的开始配体刺激。 </li><li>点击开始按钮，图像中的3D所需的时间和频率。 </li><li>在显微镜下完成工作之前，收集了Z堆栈分析过程中背景扣除以后使用。在光子计数模式，这是不必要的，因为背景基本上是0。照明接通和所有其他的成像条件相同，但与没有样品取一个样本图像。见背景减法（下）下分析。 </li></ol></li></ol>第五天 5.图像分析<ol><li> 导出图像为TIFF格式。 <ol><li>右键单击图像文件名，看菜单，然后选择导出或者单击文件|出口|进口。 </li><li>导出到TIFF为后续分析并确保出口RAW，12位数据[不是"红色蓝色绿色&#39;用于出版质量的图像（RGB）转换。不要导出为JPEG格式。 </li><li>注意在X，Y，Z的体素的尺寸和图像分析过程中的z步长，供以后使用。例如，使用一个40X / 1.4。 NA物镜这些值可能是0.138微米×0.138微米与1微米的z步长（见代表性的结果部分数据）。 </li></ol></li><li> &lt;STRONG&gt;创建分析图像堆栈。 <ol><li>打开图像定量软件（ 图2） </li><li> "从选择的序列图像行动| |新建"来从进口单序列（ 图2，箭头＃1）单个文件图像时使用。 </li><li>输入信道的数量，z轴部的数量，以及时间点的数量。 </li><li>取决于* .tiff文件中，选择的结构中，例如，通道，z平面，和时间点作为图像平面的顺序。检查最终图像堆栈正确地表示颜色通道，Z堆栈，和原来的图像的时间点。 </li><li>选择图像名称（ 图2，突出显示的文件名左，箭头＃2）。右键单击图像和/或像场，并从列表底部的选项中选择"属性"。在（X）（Y）和（Z）的大小微米像素，输入正确的图片属性上面提到。 </li></ol></li><li> 图像分析之前扣除背景。背景校正可以在任何图像分析软件来实现。扣除背景使用两个选项之一。 <ol><li>选项1:选择"操作|新建|背景减法"（ 图2，箭头＃1）。在使用上述的"暗参考"图像成像会议结束时取得的被购买背景图像下的"工具|减去背景"。 </li><li>选项2:检查在没有样品图像中的最暗的区域，找到像素的平均值或最大亮度在该地区，忽略任何异常明亮的像素，可能是虚假的荧光，随机明亮的像素，或者一些其他的现象。选择"操作|新建|背景减法"。使用该像素的亮度（由负）作为补偿。 </li></ol></li><li> 检查彩色登记，如有必要，正确的。 <ol><l我&gt;使用阳性对照，例如成像的微珠，或，共定位的囊泡和荧光染料含有GFP-（ 图3）通过切换红色或绿色通道和关闭评估用于校正的需要。在视觉上，如果登记是关闭的，即使1个像素，这是显而易见的眼睛。 </li><li>通过选择生成配准校正"操作|新建|注册校正"（ 图2，箭头＃1），并使用对话框进行调整。建成后的新文件夹的项目将出现一个标题为"注册校正"。 </li><li>通过点击图像名称申请登记到一个或多个所关注的图像（ 图2，突出显示的文件名在左侧，箭头＃2），然后选择"工具|正确注册"（工具邻近于操作按钮）。 </li></ol></li><li> 创建测量序列来分析在1A R-GFP囊泡和全细胞强度以下与血管紧张素II治疗。 <ol><li>选择一个图像（ 图2，突出显示的文件名左，箭头＃2）使用自由泳区域工具（ 图2，箭头3＃），并绘制感兴趣区域（ROI）的区域围绕一个单元格。使用扩展对焦模式（2D，下拉上左上角菜单）观察细胞的裁剪工具只在二维的可视化工作。 </li><li>右键点击图片并选择"裁剪以选择"，并产生一个新的形象的项目。 </li><li>通过点击其名称选择裁剪单元格，然后选择上面的"测量"选项卡（ 图2，箭头＃4）。 </li><li>要定义和衡量内在囊泡的GFP，在"发现"（ 图2，箭头＃5）成序列对话框（ 图2，箭头＃6）拖"找对象"。 </li><li>设置"找对象"通道到通道GFP。 </li><li>打开设置（对话框轮图标，箭头＃6），并设置4，使用阈值:"到SD和"下限"，以6集"最小对象大小"为0微米3。 注:SD下限，将取决于个人的实验。目视确认了正确的对象被阈值化是通过检查不同的时间点（ 图2，箭头＃7）制成。 "测量|反馈选项"对话框用于选择选定阈值对象的可视化的方式（ 图2，箭头＃8）。 </li><li>在"找对象"对话框中，单击"测试"（ 图2的箭头＃6），选择测量通道和类型。通常情况下，选择"所有通道"和"强度和体积的测量。" </li><li>定义阈值和过滤器对整个小区的识别和测量总的GFP强度，则重复上述步骤（5.5.4 - 5.5.8）使用以下参数:SD 0和"最小对象大小"到2μ;米3的第二个"找对象"对话框，以确定使用GFP的细胞（ 图2，箭头＃9） </li><li>在"过滤"拖"过滤人口"根据人口2序列框（第二个"找对象"对话框， 如图2，箭头＃9）。选择"卷（微米3&gt; 100）。 </li><li>视觉上确保只有一个对象被识别。在整个小区应阈值处理和其他物品过滤掉。如果该细胞是小的，例如"过滤器"（多个）参数可能需要调整。 </li><li>一旦测量已标准化，通过右键保存协议点击图像，并以重用选择"保存协议"（"协议还原"）。该协议还可以导出在相同的子目录中的图像提供了一个纪录。 </li><li>继续选择"进行测量"，"测量|设为测量项目"（ 如图2 </s仲&gt;，箭头＃8）或右键单击图片并选择"进行测量"和"所有时间点"。 </li><li>输入正确的测量名称（从图像复制粘贴方便），并分析代码或日期（一个有用的除了记录保存）并选择"所有时间点"。一个新的工作表项将图像下方创建。保存文件名简洁。 </li></ol></li><li> 分析并导出数据。 <ol><li>单击工作表的文件名，然后选择"分析"选项卡。确定是否饱和像素存在，首先选择"限制分析:"选择人口2（小区）。 </li><li>右键单击"分析标签"下，并在"分析这些数据:"在对话框中的字段中选择"MAX（[GFP颜色通道]）"; "通过概括为:"下选择"平均值（[GFP颜色通道]";并在"通过组织数据:"和"行"，选择"时间点"和CLICK "OK"。 注意:如果数据包含饱和的像素（ 例如 65536对于16位或4096为12位），该分析将是不准确的（它会低估囊泡内容）。无视分析认为细胞。如果没有饱和，继续下一步。 </li><li>其次，"原始"选项卡下，然后在"分析"选项卡，然后用鼠标右键单击该数据找到"限制分析:"下再选择人口1囊泡或人口2整个细胞。 </li><li>下"分析这些数据:"选择"总和"（GFP颜色通道）;在"通过概括为:"选择"总和";和"行"，选择"时间点"或"相对时间"，然后单击"确定":并在"通过组织数据"。分析选项可以保存并重复使用。 </li><li>选择和复制/直接从图像定量软件粘贴到空白电子表格或通过右键导出电子表格数据点击DAT和选择保存为.csv文件。全细胞强度测量将包含群体中的每个囊泡的测量，所以一定要识别和它从囊泡分离。 注:绘制数据后，包含小泡内的总GFP荧光的增加急剧上升。光漂白，如果发生了，被检测为随时间的总细胞的GFP强度的损失。 </li></ol></li><li> 创建测量序列来分析目前在溶酶体GFP。 <ol><li>在查找对象（人口1），设置"查找对象"通道到红色通道和6（ 图2）设定的"阈值使用:"由箭头＃表明对话框内轮图标SD和"下限"到6.设置"最小对象大小"到0.017微米3。 注:SD下限将取决于个人的实验。正在阈值处理应该由考试作出正确的对象视觉确认进不去不同的时间点。 "测量|反馈选项"对话框用于选择选定阈值对象的可视化的方式（ 图2，箭头＃8）。 </li><li>在"查找"对象单击"测量""对话框（ 如图2所示的箭头＃6），选择测量通道和类型。通常情况下，"所有通道"和"强度和体积测量"被选中。 </li><li>人口2，等同于使用设置用于识别表达在1A R-GFP（5.5.9）整染细胞。 </li><li>如果多个细胞（包括未转染的细胞用荧光染料阳性囊泡）都存在，在分析的第一阶段裁剪细胞是至关重要的。可替代地，使用"划分"应该被用来限制表达在1A的R-GFP转染的细胞内的溶酶体鉴定。溶酶体必须仅GFP的细胞内被识别和不从附近的，未转染细胞。 </li><li>如上分析和导出数据。 注:阈值处理的细胞和小泡的一个例子示于图4。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

这里介绍的初步实验表明，在24分钟相当短的时间过程，在交付在1A R-GFP到溶酶体没有明显的变化发生了。一般来说，最早可能在15-20分钟出现交货内在受体溶酶体。这个实验是用的内化在1A R的大部分是针对大，核周内涵体的假设是一致的。证据表明，至少一些在1Ař到达溶酶体由Li 等人证实。 ，谁相比在血管紧张素II治疗后5分钟和30分钟的细胞，并发现与在1AřLAMP1的显著共定位于30而不是5分钟6。 本文提供细胞制备，成像和分析步骤的详细说明，并讨论了这一先进成像技术的许多固有的缺陷。这种方法依赖于下面讨论的关键步骤。 由于细胞的身体健康为任何活细胞成像实验的关键，是要避免使用，因为在生长速度和更高代的转染效率变化的细胞外通道11是很重要的。为了保持整个实验过程中的健康细胞中，由于脂质体溶液是有毒的通过增加膜起泡证明细胞的转染混合物与细胞温育期不应超过5个小时。温度，湿度和CO 2控制是之前以及在成像期间显著。在成像期间在温度漂移可导致相对大的z部分的漂移和的焦平面稳定性损失。因此除了血管紧张素Ⅱ的至细胞成像期间中建议额外小心，以确保焦平面被准确地维持。 成像使用固定的细胞在免疫染色的活细胞成像的优点是该体内条件可以严密地控制并能避免与固定的细胞相关的伪影。但是，活细胞成像是具有挑战性的，以优化是非常迅速，例如受体内在细胞过程。仪速度和需要避免漂白限值可以研究事件的选择。每个成像实验可能要求某些选择在某些参数和妥协他人进行;实例是在像素大小，行累积，缩放因子，和扫描速度的选择，以得到用于分析的良好的质量的数据，并避免过采样在同一时间。在这些因素的关键决定取决于平衡用于获得适当的像素大小来区分个体囊泡以及需要用于获得时发生的事件的足够的时间分辨率的需要。因此，一些权衡必须以获取信息的速度不够快的细胞开始内化GFP标记受体制成。在我们的情况下，尽管现代共聚焦软件可以建议的最佳像素尺寸，我们的实验中使用小于根据奈奎斯特等式14的最佳像素大小的值。的其他实验设置最佳的选择也将取决于细胞如何亮是因为较大的像素尺寸将使光子允许折衷较长体素的停留时间的快堆积以收集更多的光子。 为获得成功的分析中，活细胞成像也极为需要的时间推移成像期间精确焦点稳定化。焦移并未能像整个小区可以在受体内化的量化深远的影响，以及细胞总GFP强度。过程中加入血管紧张素Ⅱ的下列成像最早时间点，焦点偏移的这个问题的解决方案已通过共聚焦厂家在自动对焦溶液的形式被提供，由此盖玻片interferometry利用IR激光器加上反馈到自动聚焦控制用于从目标的相对距离保持于由IR反射标识盖玻片表面（ 例如正定聚焦或自适应聚焦控制）。该显微镜的其余问题是再因此尽管形状的变化，整个细胞保持在成像体积来识别正确的z范围。温度保持在另外血管紧张素Ⅱ的初始后的时间点对焦/ Z位置的另一个关键因素。物镜的温度以及活细胞成像室应在整个实验中保持在37℃。 成像的另一个重要组成部分，是定量地采集图像。如果一个路政署探测器在光子计数模式用的摄像参数必须被设置为使得光子堆积不会发生。对于光电倍增管和其他探测器，图像不得含有饱和像素（体素）。在这两种情况下，一个SPEcialized查找表（LUT）可用于可视化的饱和像素的，当然，如果它们存在的存在下，在显微镜应减少激光的强度和/或增益。 分析尽管这里描述的方法被施加到在1Ař受体及其与溶酶体的共定位，这是适用于实验，其中用各种荧光标记的亚细胞区室受体的相对量是在问题。如果目标是衡量是否受体移动到细胞器，然后使用Pearson的共定位系数与用于细胞器标记物是有问题的。其中细胞器被识别的每个像素将具有标记物的高量，以使系数之一永远是接近100％。因此，共定位测量可能会产生误导。另一方面，测量受体的百分比的细胞器与雷杰普总量器是更丰富和更清晰的解释。其中蛋白质的共定位可能使用最好的Pearson相关系数来描述一个很好的例子是在1A受体- [R LAMP1和其互动已经被记录在案，尽管通过更复杂的共定位的方法，即FLIM / FRET 6。 利用三维成像比使用，因为时间过程（ 实时 成像2电影 ）中是不可避免的细胞运动的二维与分析细胞的代表性切片更准确。它是由在图中所示的活细胞的图像清晰3，图4B和图5A，该受体主要定位于质膜，但是，暴露于血管紧张素II几分钟它定位于在膜和小亮点内囊泡相邻的膜，可能有被小窝和早期内涵体（ 图4B </strong&gt;的信息，4℃）。随着时间继续发展，囊泡通过细胞移动积聚在邻近该已经被描述为多泡内体细胞核大囊泡（MVEs， 图1，图5A）3，15。通过细胞随着时间的推移将扭曲的内化过程和歪曲的真实事件特别是如果片不包括在早期细胞表面，并在稍后的时间MVEs单个片。 在这项研究中，用于图像分析两种方法可以最大限度地减少对定量结果的GFP漂白以及荧光染料的影响。作为细胞光漂白（虽然在有限的程度），相对于整个小区强度内在小泡的相对亮度被保持，假定漂白发生均匀。荧光染料阳性囊泡也容易通过选择标准deviatio识别对于阈值亮内在囊泡平均荧光强度选项的n个。从这个背景甚至荧光下跌的整体水平区分开来。因此，我们的测量结果不温和漂白的影响敏感。然而，随着时间的推移细胞强度的强漂白例如50％的损失可能具有在定量结果的影响更大。可替代地，光漂白的效果可以测量独立地在对照实验中不存在血管紧张素II中在1A的R-GFP的情况下，或在荧光染料的情况下，相邻的未转染细胞。漂白的影响可能再分析过程中被分解英寸其他问题最近，细胞生物学家已经进行荧光蛋白的严格的评估和修改用于监测膜贩卖细胞，以避免由荧光蛋白本身生成的工件，独立于蛋白质拜因摹标记。各种荧光蛋白易受与局部微环境导致蛋白质沉淀或基于它们的pKa值和氨基酸半胱氨酸的存在下，分别为16的交联的相互作用。这些相互作用的另一个结果可包括荧光16的损失。所有这些文物的影响贩卖定量测量。康斯坦丁尼等。已经产生用于成像的单体荧光蛋白缺乏半胱氨酸，以消除潜在的工件16。因而，取决于成像实验的目标，从EGFP（非单体）切换到一个单体的，自由半胱氨酸的形式将是酸性和/或减少水疱微环境的研究是有利的。另一个例子是其改善了FRAP效用或FRET实验，其中多聚和交联将影响蛋白扩散和蛋白质 - 蛋白质相互作用。 <p类="jove_content"&gt;一个附加的条件是，它测试EGFP标签是否修改了受体的正常行为是很重要的。测试这种可能性的一种方法是通过确定是否存在由进行关于与野生型或EGFP标记的受体转染的细胞的放射性配体结合测定中受体亲和性（Kd值）和最大的受体数（Bmax的）的任何差异。 另一个潜在的神器考虑使用起来红LysoTracker与GFP融合蛋白成像溶酶体当是LysoTracker红已被证明能够光转化的绿色17。笔者建议使用落射荧光照明的最小化，讨论这个神器的出现，并使用此试剂是指共存的成功实例。此外，溶酶体其他标记也可以被用来验证共定位的结果，例如，标记LAMP1 6。 今后，该测定的灵敏度和特异性可以通过使用巴弗洛霉素阻止降解但不积累7抑制溶酶体酸化，18待检查受体通过使用小泡转运和/或融合的抑制剂靶向溶酶体以及其它亚细胞区室通过使用氯喹来阻止与溶酶体因此阻断受体与溶酶体2，5，6，7，18的共定位，通过使用阳性对照，例如标记的血管紧张素Ⅱ的溶酶体2靶向融合，并通过比较其它公-described受体与配体系统，例如转铁蛋白或表皮生长因子和它们的同源受体3。一个有趣的阳性对照，一lbeit假象，在我们的实验是在相邻的，未转染的观察，在1A的R-GFP阴性细胞的结果。这些细胞显然拿起从转染细胞释放GFP和它靶向溶酶体，其中共定位用荧光染料是相当突出（未示出）。这种共定位，但是，由于预期并不是血管紧张素II敏感。 总之，这里提出了转染，成像和图像分析的方法提供了通过该受体内在各种亚细胞区室可以在活细胞在一段时间内定量地跟踪的装置。不同的受体或选择性突变受体之间的相对比较是可能的关于动力学和亚细胞定位至特定区室。可能遇到的困难，并与该技术相关的文物进行了讨论。尽管如此，加上图像分析快速实时成像在3D可用于测量受体摄取和快速通过细胞质转移到分子定义的车厢。此设置阶段为从受体或受体激动剂，拮抗剂和抑制剂的应用突变产生的差异测量。

总体目标是为了获得定量证据在时间和与具体的亚细胞器的受体共定位的空间与受体激动剂治疗后。因此，这里的当前的目标是捕获溶酶体时间推移共焦图象和一个跨膜在人胚肾细胞转染和随后收集和分析在三维成像数据定量测量到输送受体受体（HEK）溶酶体。通过受体配体激活可以帮助确定跨膜受体是如何生理和病理条件下的监管调查时与溶酶体或其他细胞器跨膜受体的内吞作用的过程中同时进行贩卖。 在1A R被假定在溶酶体中被降解以下与血管紧张素II治疗细胞模型系统，主要HEK293 1虽然大多数的RECeptors被主要定位在多泡循环核内体购买回收到质膜而配体，血管紧张素II，大头是在溶酶体2，3，4，5劣化。最近，Li 等。使用荧光共振能量转移（FRET）和荧光寿命成像显微术（FLIM）技术显示，在1Ař共定位（在〜10纳米尺度）与LAMP1，溶酶体膜蛋白表明至少一些受体靶向于与劣化被溶酶体6。这些作者指出，溶酶体抑制剂氯喹阻止在1A的R-LAMP1关联这与以前的报告表明氯喹的效果是阻止晚期内涵体或自噬体与溶酶体融合一致。其他间接的方法来确定在1A中R 1ocalization在溶酶体已经利用巴弗洛霉素，溶酶体抑制剂来推断AT在溶酶体3，4，5，6，7，8 1a中 R存在。 活细胞成像避免了细胞体积，和GFP-嵌合受体的损失/调光/再分配上固定的潜在影响。在固定的细胞先前的研究已经依靠确定共定位或受体与溶酶体或使用标记的受体结合替代物如活细胞共现β-arrestin的1，2，3，4，5，6。使用旋转盘共聚焦或激光点扫描的conf其他GPCR的活细胞成像配备的GaAsP或路政署探测器OCAL显微镜使研究者观察到在30秒的时间间隔9，10 Z-栈成像单个细胞受体的内化和贩运。但是，即使当活细胞的z堆栈迅速收集，可能每个堆叠， 即在2D 10内的单一平面的选择之后才发生分析。虽然这可能在问题研究内化的GPCR或酪氨酸激酶受体是足够的，则在1A卢比积聚在该改变的大小和位置随着时间的推移，因此在本质上更难以用一个代表性的单个片在每个时间点能充分检验囊泡。彻底确定所述标记的AT 1A R至细胞并检测囊泡在尺寸和位置不同的各亚群的途径，我们已经设计出用于3D成像和三维分析方法来跟踪这些变化，并为<html>在与各种细胞内隔室的标记一起使用，如溶酶体。 调查人员可以使用该协议为活细胞成像可视化直接在1A R的运动进入细胞和它下面的血管紧张素Ⅱ刺激转移到细胞内车厢。亚细胞标记有荧光蛋白嵌合体或其他荧光标记。该协议也可以用来作为第一种方法本地化与200nm的最小分辨率亚细胞器的受体以比较突变与野生型受体或检测以下药物治疗的变化。该技术是可访问的，因为它可以在配备用于活细胞成像的任何共聚焦显微镜下进行。相对容易这种方法具有增加的专业知识，并进行其检测分子间的相互作用6 FRET / FLIM / BRET技术所需的设备形成对比，"外部参照"&gt; 11。这些测量确定在高分辨率（〜10纳米）和亚细胞器内的定位推断蛋白 - 蛋白相互作用。这些更先进的技术用于跟进并进一步定义感兴趣分子间的相互作用，而不是受体通过亚细胞区室的通道，和它们直接证明了电池11内的特定的时间和地点的蛋白质-蛋白质相互作用。 FLIM，FRET一个独立的技术受主浓度的，是在固定的样品，因为图像采集速度慢最常进行的。相比之下，比FRET提供快速成像和蛋白质相互作用的高分辨率共存。比率的FRET的缺点是，成像利用宽视场落射荧光来获得成像速度和包括整个细胞，导致降低的分辨率和细胞器12的对比度。生物发光共振能量转移（BRET）是另一个已经应用到的GPCR来测量采集分子接近的随时间11先进技术。在这种技术中的蛋白荧光团分子分开并每一半连接到所讨论的两种蛋白质之一。当利益绑定的两种蛋白互相嵌合标签的零件重新组装获得的荧光随着时间的推移定量增加荧光。 在这里，我们提出了结合图像定量研究在1A的R贩运在血管紧张素Ⅱ刺激和交付的内化在1A R以下列血管紧张素Ⅱ刺激溶酶体的电位变化的细胞活细胞成像一个简单的技术。这种技术的最终用途是表征膜贩运野生比较型和突变受体的差异。这些差异将有助于确定该机制（S）的影响，在1A - [R leadi的生理活动ng到调节血压。

<table><tbody><tr><td>Angiotensin II (Ang II)</td><td>Sigma-Aldrich</td><td>A9525&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)</td><td>Gibco -ThermoFisher Scientific</td><td>11960-044</td><td>Warm in 37 &amp;deg;C water bath before use&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F2442</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Gibco -ThermoFisher Scientific</td><td>15140-122</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>Gibco (life technologies)</td><td>25030-081</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Human Embryonic Kidney (HEK)-293 cells</td><td>American Type Culture Collection (ATCC)&amp;nbsp;</td><td>CRL-1573</td><td>Passage number 5 - 12</td></tr><tr><td>Nunc Lab-Tek II chambered 1.5 German Coverglass System</td><td>Sigma-Aldrich</td><td>155409</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>11668019</td><td>Store at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Gibco- ThermoFisher Scientific</td><td>31985-070</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>PBS 10x&amp;nbsp;</td><td>Fisher BioReagents</td><td>BP3994</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Dulbecco&#039;s Modified Eagle medium (DMEM 1x)&amp;nbsp;</td><td>Gibco -ThermoFisher Scientific</td><td>31053-028</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>LysoTracker Red DND-99</td><td>molecular probes -ThermoFisher Scientific</td><td>L7528</td><td>Store aliquotes in dark at -20 &amp;deg;C .&amp;nbsp;</td></tr><tr><td>Volocity Ver. 6.3 Image Analysis software</td><td>PerkinElmer</td><td>Visualization and Quantitation Modules</td><td>For 3D image analysis of image stacks</td></tr><tr><td>Leica SP8 AOBS</td><td>Leica Microsystems</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr><tr><td>Zeiss LSM 880</td><td>Carl Zeiss</td><td>laser scanning confocal microscope</td><td>For image collection</td></tr></tbody></table>

live cell imaging and 3d analysis of angiotensin receptor type 1a trafficking in transfected human embryonic kidney cells using confocal microscopy

活细胞成像是用于同时捕获在转染人胚胎肾293（HEK）细胞在用血管紧张素II（ANG II）的刺激血管紧张素1a型受体（在1A R）和胞内隔室的时间推移的图像。 HEK细胞中瞬时与含有具有标记增强型绿色荧光蛋白（EGFP）1A耐药质粒DNA转。溶酶体标识用红色荧光染料。活细胞图像捕获对血管紧张素II刺激后激光扫描共聚焦显微镜和三维（3D，体素），随着时间的推移通过软件进行分析。活细胞成像使调查受体贩运和避免了与固定相关的困惑，特别是，绿色荧光蛋白标记的膜受体的损失或人工的位移。因此，作为单独的细胞通过时间跟踪，受体的亚细胞定位可以被成像和测量。图片必须获得sufficientlŸ迅速捕捉快速运动的囊泡。然而，以更快的成像速度，收集光子的数目被减小。妥协还必须在成像参数，如体素尺寸的选择，以获得成像速度进行。活细胞成像显著应用对蛋白质运输，迁移，增殖，细胞周期，细胞凋亡，自噬和蛋白质 - 蛋白质相互作用和动态，学习到仅举几例。

在该代表性组实验中，HEK细胞用克隆到质粒pEGFP-N2质粒13血管紧张素1a型受体（在1A的R-GFP）的构造（E1,2,3-在1A R）的转染。 HEK的延时图像被收购，发挥作为一个电影（见活细胞成像1和活细胞成像2影片 ）。电影，静止图像显示，配体刺激后，在1A的R-EGFPs都主要在质膜局部的，但加入血管紧张素Ⅱ的这些受体成为内在化2.5分钟内以形成明亮的囊泡和在稍后时间群集在较大的囊泡核周区（ 图4B，5和6A;活细胞成像2）。 在图5中P TOTAL细胞的GFP强度和总囊泡的GFP强度用于小区的量化rovide并发症固有时血管紧张素Ⅱ诱导皱裂及细胞形态的变化紧跟刺激的图示（ 图5A，在0分钟后血管紧张素Ⅱ箭头，上排，橙色表示阈值的对象）。在稍后的时间，含GFP-泡在核周群定位也干扰（ 图5A，红色箭头为29.5分钟）。虽然所有对象都应该被包括在总细胞的GFP强度测量，所述褶边应当从囊泡测量，因为它们代表质膜在1A的R-GFP的排除。相反，聚集核周囊泡不应从囊泡测量中排除（后者在包封在图5A中红色圆圈的小区的一个区域中可见，在29.5分钟箭头后血管紧张素II，下排）。试图通过大小筛选来完成此（大于或小于7微米3）是没有用的，因为它不能区分褶边和集群vesicles（ 图5A和5B，比较过滤&gt; 7，过滤器&lt;7，且没有过滤器）。另一种可能是制定感兴趣区域（ROI）的地区涵盖了细胞的细胞质部分，但不包括电池的边缘皱裂和周长。最终，该小区将不被包括在定量结果，因为有分别的饱和本GFP的像素。 第二个有代表性的例子示于图6A中示出，其中，不使用自动聚焦的时间过程。内化GFP被示为百分之总细胞的GFP（ 图6B）。在3分钟的红色箭头前的时间点是在误导细胞，因为只有部分是在成像体积，但血管紧张素II加入后3分钟，焦平面再次稳定。在细胞内的总的GFP表明该细胞不随时间明显光漂白（ 图6C<html> ）。 3至约12分钟，在泡增大后成水平（ 图6B），百分比GFP强度。接着，通过荧光染料标识溶酶体被用来确定一个转染的细胞然后将其的GFP强度（ 图6D）的总和量化内的ROI。三分钟后在1A的R-GFP的红色标题溶酶体的强度不随时间变化（ 图3D中，n = 5，平均均值±标准误差）。因此，有在分娩在1A R-两性平等问题协调到溶酶体没有检测到增加对血管紧张素Ⅱ给药后长达24分钟。 <img alt="图1" src="/files/ftp_upload/55177/55177fig1.jpg" /> 图1:饱和像素的查找表（LUT）来识别。红色箭头表示以蓝色显示饱和像素。T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55177/55177fig2.jpg" /> 图2:图像量化软件概述。关键项都用数字表示，并在方法文本中讨论。 1）行动项目，2）选择图像，3）手绘ROI工具，4）测量标签，5）找对象，6）人口1找对象对话框中，7）箭头激活时间的推移电影，8）测量项目， 9）第二个找对象包含过滤人口命令对话框。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/55177fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55177/55177fig3.jpg" /> 图3:颜色注册校正。与裸眼图像和校正后的像素登记用含有在1A的R-GFP几个colocalizing囊泡插图一起示。红色像素错开1个像素的左侧的未校正图像的XY平面。插图显示在更高的放大倍率盒装地区。比例尺= 3.3微米，像素尺寸= 0.138点¯x0.138×1微米3。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/55177fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55177/55177fig4.jpg" /> 图4:阈值的插图识别GFP-和含染料荧光囊泡和GFP-整个细胞。 （A）使用在1A的R-GFP（绿色荧光蛋白，顶行）的全细胞的鉴定和阈值处理的GFP整个小区（ID细胞，底行），在0被示出和血管紧张素II加入后2.5分钟。白框表示插件在（B）和（C）所示等领域。 （B）在1A的R-两性平等问题协调在血管紧张素II此外在单色图像（顶行）后0和2 5分钟进行比较。通过阈值确定的小泡显示在最后一行（ID新村，紫）英寸（C）两彩色图像（在1A R-GFP /荧光染料）在0和2.5分钟后血管紧张素II除了所示。通过阈值确定溶酶体用红色轮廓显示。比例尺= 7.0微米，体素尺寸= 0.114点¯x0.114×1.4微米3。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/55177fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55177/55177fig5.jpg" /> 图5: 褶边和集群囊泡对内部化GFP受体的测量影响的插图。 （A）<html> Ť1a中的R-GFP在血管紧张素II处理的细胞，在0（上排）和2.5分钟（底行）示于其中在第一列中的过滤器只适用于选择的对象大于7微米3三种过滤实验中，在第二列以选择对象少7微米3，并且在第三列不使用过滤器。阈值水平在所有三个不变。箭头指示大阈值的对象，圆圈表示含有的核周囊泡在稍后的时间点细胞质的面积。阈值区域是橙色。 （B）在囊泡GFP总强度的量化显示为带或不带大小过滤（按列A）在两个时间点（由行A）阈值的区域。像素尺寸为0.138 0.138点¯x×1微米3。比例尺= 5.6微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/55177fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ntent"FO:保together.within页="1"&gt; <img alt="图6" src="/files/ftp_upload/55177/55177fig6.jpg" /> 图6:跟踪AT1 一个 R- GFP内在潜力和本土化溶酶体。 （ 一 ）在1A R-GFP与LysoTracker红五选定的时间点，从伴随电影（活细胞成像1片）为0，3，6，9，12分钟后血管紧张素Ⅱ所示。网格表示的6.56微米3单元规模，体素尺寸为0.297点¯x0.297×1.2微米3，并且沿z步骤= 1.2微米。 （B）在小泡随着时间的推移％的总的GFP的曲线图示出了在1A的R-GFP的快速内在化。 （C）在细胞随着时间的推移，总GFP的情节显示，在成像的24分钟发生相对较少的漂白。 （D）的溶酶体被识别LysoTracker红阳性囊泡鉴定。佛罗里达州内的总GFP强度uorescent染料阳性囊泡的ROI在每个时间点（归到与光漂白不赔偿其不能独立测量的第一时间点）进行测定。 （C - D）在此之前的箭头，则测量不因为重心转移有效。 N = 5，平均值±平均值的标准误差。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/55177fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/55177/55177mov1.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/Live_Cell_Movie_1.MOV">电影1:活细胞成像1:（右键点击下载）</a>与AT 1A R-GFP转染的HEK细胞成像是每1分的加100nM的血管紧张素II后24分钟（25帧）。电影是用激光扫描共聚焦显微镜有计划，Apochrom收购在63X / 1.4 NA油浸物镜的0.279点¯x0.279×1.2微米像素3的尺寸;和5片，共计4.794微米厚度的3.20微秒/像素成像。电影率为2帧/秒。单位= 6.56微米。 <img alt="电影2" src="/files/ftp_upload/55177/55177mov2.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55177/Live_Cell_Movie_2.mp4">电影2:活细胞成像2:（右键点击下载）</a>与AT 1A R-GFP转染的HEK细胞，每隔30秒拍摄的另外100纳米血管紧张素Ⅱ的25分钟后（51帧）。电影是用激光扫描共聚焦显微镜有计划复消色差透镜40X / 1.4 NA油浸物镜收购。像素尺寸为0.11点¯x0.11×1.4微米3。 7片共计8.4微米厚的在3.72秒/帧进行成像。电影速率为3帧/秒。比例尺= 7微米。

Watch this Scientific Journal Video about Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy at JoVE.co

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

在这里，我们提出了一个协议，以表达绿色荧光蛋白标记的血管紧张素1a型内吞过程中血管紧张素Ⅱ开始治疗受体细胞图像。这种技术包括用第二荧光标记物标记的溶酶体，然后利用软件随时间来分析受体和溶酶体的共定位在三维空间中。

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

live-cell-imaging-3d-analysis-angiotensin-receptor-type-1a

Universidad San Sebastian

Research

JoVE Journal

Biology

9.6K 次观看.  Georgetown University Medical Center. 在这里，我们提出了一个协议，以表达绿色荧光蛋白标记的血管紧张素1a型内吞过程中血管紧张素Ⅱ开始治疗受体细胞图像。这种技术包括用第二荧光标记物标记的溶酶体，然后利用软件随时间来分析受体和溶酶体的共定位在三维空间中。 

视频: Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

生物工程

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

发育生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

生物学

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

神经科学

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

追踪药物诱导Colocalizational变化分析受体后内部贩运

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...