This work is supported by the intramural fund of NIAID, NIH.

1.文化和中性粒细胞样人类分化HL60细胞<ol><li>制备RPMI（罗斯韦尔园区纪念研究所）1640培养基含有RPMI 1640培养基，10％（体积/体积）胎牛血清（FBS），0.1mM的丙酮酸钠，和25mM HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸）。暖RPMI 1640培养基至37℃。 </li><li>使用血球确定HL60细胞的细胞密度，以确保细胞在对数相增长阶段。 注:细胞通常是在传代后的第三天数期。数期细胞的密度通常为6-8×10 5个细胞/ ml。开始新的培养所需的细胞密度为约1.5×10 5个细胞/ ml。 注意:例如，在T-75扁平烧瓶10ml的新培养，如果数期细胞的细胞密度为6×10 5个细胞/ ml，然后2.5对数期的细胞（毫升6×10 5个 ）稀释到1.5×10 5细胞/ ml的10毫升L NEW文化。打10毫升培养的终体积，7.5毫升RPMI 1640培养基中加入。 </li><li>放置暖RPMI 1640培养基的计算量成扁平烧瓶中。对于T-75扁平瓶培养，细胞培养物的总体积为10毫升</li><li>添加数期生长的HL60细胞的计算量成扁平烧瓶达到1.5×10 5个细胞/ ml的细胞密度。 </li><li>孵育在37℃下将细胞在5％的CO 2的潮湿培养箱中。 注:HL60的倍增时间几乎是24小时。 </li><li>通道中的细胞用RPMI 1640培养基每2-3天。重要的是，在HL60细胞的细胞密度不超过1.5×10 6个细胞/ ml和在孔通道持续超过2个月。 </li><li>实验前，区分HL60细胞5天。 注:RPMI 1640分化培养基含有RPMI 1640培养基，10％（体积/体积）胎牛血清（FBS），0.1mM的丙酮酸钠，25毫米高EPES，和1.3％二甲亚砜（DMSO）中。换句话说，它是在RPMI 1640培养基1.3％DMSO中。 <ol><li>分化的HL60细胞，预暖RPMI 1640培养基至37℃。温暖的RPMI 1640培养基加入到一个平坦烧瓶中。 </li><li>要最终10毫升HL60分化培养，直接在平坦烧瓶添加130微升DMSO中的RPMI 1640培养基而回旋烧瓶，使DMSO的迅速被RPMI 1640培养基稀释。 </li><li>添加数期HL60细胞以1.5×10 5细胞/ ml的终细胞密度在最后的10毫升的RPMI 1640分化培养基。培养HL60细胞在RPMI 1640分化培养基中在37℃在5％的CO 2的潮湿培养箱中5天。 </li></ol></li></ol> 2.涂层4well室的外盖玻璃表面<ol><li>取的2％明胶原液瓶子从冰箱，用70％乙醇清洗，并将其放置在引擎盖。 ŧAKE1毫升2％明胶原液并立即返回剩余的明胶原液的冰箱。 </li><li>允许明胶溶液完全在37℃下液化，并彻底地吸取​​它。稀释的2％的明胶原液用9ml预温1×Hanks平衡盐溶液（HBSS）缓冲液，以0.2％明胶的终浓度。 </li><li>在HBSS中加入0.5 ml或2毫升0.2％明胶至4孔腔室的两个中间井或1-井室在底部的盖玻璃，分别。倾斜在几个方向的板，以使液体覆盖整个表面区域。 </li><li>将平板在37℃培养箱。板将准备在1小时内使用。它们可用于长达5天。刚刚播种细胞前，请从4孔或1井室的明胶溶液。 </li></ol> 3.趋化实验使用细胞运动分析装置<ol><li>准备和预热RPMI 1640挨饿介质，它是RPMI含0.1丙酮酸钠（可选）和25毫米的HEPES网上平台。 </li><li>在RPMI 1640准备100微升100纳米fMLP的（N-formylmethionyl亮氨酰苯丙氨酸）的饥饿中。 </li><li>制备3-毫升1-％牛血清白蛋白的（BSA）/ RPMI1640培养基，其中包含在RPMI 1640 1％BSA的饥饿培养基和10毫升0.1％BSA / RPMI 1640培养基。 </li><li>外套22平方毫米盖玻片并用新鲜制备的1％BSA的一个5微米的细胞迁移分析装置芯片/ RPMI1640培养基在RT 1小时。 </li><li>组装持有人使用下列步骤: <ol><li>使得杆可以朝向用户倾斜与垂直位置的杠杆支架底座的位置。应用70％的乙醇溶液，以41毫米的玻璃的表面上。仔细擦拭了擦拭表面，小心不要刮伤它们。 </li><li>插入41毫米的玻璃插入支架底座。放的BSA包被的22毫米正方形盖玻片上41毫米的玻璃在保持器基座的顶部。 注:涂コ41毫米的玻璃和芯片之间overslip允许趋向于涂层表面的基质发生。 </li><li>插入O形环（小）进入晶片外壳的底部，并在基片壳体安装到与在后的椭圆孔的支架基座。向前拉保持器基部的内部水平至水平位置锁定在适当位置的基片壳体。 </li><li>从与空气除尘器晶片壳体的内部吹出的灰尘。加入4毫升0.1N％BSA / RPMI 1640培养基到晶片壳体。 </li><li>安装的BSA包被的细胞迁移分析装置的芯片在晶片壳体的与结构面向下在与玻璃接触的中心。 注意:在芯片需要与在后的定位标记（在芯片的顶部的孔）插入。 </li><li>通过在橡胶密封垫插入孔的两个凸部嵌合贴上橡胶垫圈到晶片夹具的底部。 </li><li>安装O型圈（大）到薄片壳体的顶部。安装在后部传感器孔的晶片把持。向前拉壳​​体基部的外侧杠杆到水平位置以锁定该晶片夹持在适当位置。 </li><li>组装支架后，放置在光盒的保持器的底部，以确认有在孔中没有气泡。如果检测到气泡，利用所提供的塑料注射器装有一个样品装载尖端移除。 </li><li>取下盖子，并把装配到板上的电池移动分析装置单元的顶部。将传感器块在支架。 </li></ol></li><li>准备HL60细胞趋化含量: <ol><li>计算出分化的HL60细胞用血细胞计数器的细胞密度。后分化5天后，细胞密度是大约1.0×10 6个细胞/ ml。 </li><li>计算HL60细胞的最终体积以2×10 6个细胞/ ml的终细胞密度。例如，如果的细胞密度分化的HL60细胞为1.0×10 6细胞/ ml，然后在10毫升分化的HL60细胞将用5毫升0.1％BSA / RPMI 1640培养基中重新悬浮以达到2×10 6个细胞/ ml。 </li><li>离心机分化的HL60细胞在200 XG在RT 5分钟。用0.1％BSA的计算量除去上清液和重悬细胞/ RPMI在步骤3.6.2 1640培养基），以2×10 6个细胞/ ml的终细胞密度。 </li></ol></li><li>启动细胞的流动性分析装置和PC进行图像采集。将本机连接到PC。转细胞迁移分析装置上。打开电脑。 </li><li>启动了细胞分析移动设备软件。 <ol><li>观察5个控制面板:摄像头，取暖器，射击，备忘录，以及摄像机图像。 </li><li>上的摄像机图像的控制面板，用"水平线"或"垂直线"，以实时调节保持器/视图位置到中心的相机在相机图像面板。 </li><li>在相机的控制面板中，选择CH1至CH6的摄像机移动到所定义的通道。要居中通道的视场，请单击"移动L"或"移动R"调节摄像头的X坐标和水平居中摄像机的位置。 </li><li>调整通过转动细胞迁移分析装置单元的前面板上的位置旋钮垂直调整图像居中屏幕摄像机的Y坐标。 </li><li>在加热器控制面板中设置"持有人的临时"到37.0℃，"板温度"到39℃。点击"热"开始升温。同时按"持有人"控制使用连接到支架上的热传感器的温度。 </li><li>拍摄面板中，输入"15秒"的"时间间隔"和"30分钟"在"时间"的时间间隔和趋化性试验的持续时间。检查安全原因"在拍摄结束时关闭加热器"。指定日stination用于保存的图像。 </li><li>在备忘录面板，该实验的输入的细节，诸如细胞系中使用，无论是否施加治疗，并应用于化疗引诱的浓度。 </li><li>确认所有将在实验中使用的信道的中心位置，并根据需要重复该调整所有通道。 </li></ol></li><li>注射并调整单元格如下: <ol><li>取下支架上的缓冲区，以及从顶部为每个通道取出8微升缓冲从第三。 </li><li>用注射器注入2微升细胞进入第二阱中相同的信道，同时监测实时控制细胞数和注射过程中流动的屏幕。细胞有良好的对齐后，立即加入了8微升缓冲回来。重复同样的程序为其它信道（ 图1B）。 </li></ol></li><li>加入2毫升缓冲液回夹持器，并添加1μl的100nM的调频LP从顶部第三阱。启动图像采集和保存数据。分析使用图像跟踪软件9（ 图2）获得的数据。 </li></ol> 4.转染电穿孔<ol><li>分化HL60细胞5天实验前，如第1大衣4孔或1井室描述为在第2节的指导。 </li><li>制备和温暖的RPMI 1640电恢复介质，其中包含的RPMI 1640培养基，20％（V / V）FBS，0.1mM的丙酮酸钠，和25mM HEPES，至37℃。 </li><li>右开始转染实验之前，添加任一300μl的3毫升的RPMI 1640培养基成的预包被4孔或1-井室的孔中，分别。 </li><li>孵育室在湿润的37℃培养箱，用5％ 的 CO 2。立即使用或储存这些商会在孵化器，以供将来使用（两周）。 </li><li>算分化HL6的细胞密度0细胞用血细胞计数器。 注:细胞密度通常达到约1.0×10 6个细胞/ ml。细胞密度超过3.0×10 6个细胞/ ml通常给不希望的转染效率。如HL60细胞是悬浮细胞，则不需要再悬浮。 </li><li>离心细胞，在200 XG在RT 5分钟。除去上清，悬浮2×10 6 HL60细胞在100微升转染试剂。 </li><li>添加GFP-PKD1质粒9的4微克到细胞和转染试剂的100μl的混合物，并轻轻调匀。该混合物添加到提供的反应杯。 </li><li>选择制造商的预先设定的程序的人白细胞的细胞系并进行电穿孔。 注:更多信息，请从制造商的网站。 </li><li>电穿孔后，立即轻轻添加预热500微升的RPMI 6140电恢复介质向细胞，并轻轻地从反应杯与设置在塑料移液器转移细胞到1.6毫升管。 </li><li>温育30分钟将细胞在加湿37℃培养箱，用5％ 的 CO 2。种子100微升或500微升的细胞成分别一4孔中的一个孔或一个1阱室，。 </li><li> RPMI 1640培养基加入1毫升或4毫升至4孔的同样良好的终体积或1-井室，分别孵育所述细胞在加湿37℃培养箱，5％CO 2下3小时。预暖的RPMI 1640饥饿培养基至37℃。 </li><li>用1ml的移液管，轻轻地从一个4孔腔的孔或1-井室分别除去0.8毫升3毫升的RPMI 1640培养基。要轻柔，避免吸走秉承HL60细胞。 注:HL60细胞生长，并在悬浮液介质是有区别的。被分化或具有一定的治疗后，HL60细胞粘附涂有聚赖氨酸，胶原，明胶，或网络连接的底层纤连蛋白。 </li><li>添加0.8毫升3毫升RPMI 1640挨饿介质进入一个4孔或1井室的很好，分别为。等待1小时。 注意:此时的细胞准备用于成像实验9。 </li></ol> 5.监测GPCR介导通过多通道荧光显微镜PKD1的膜转<ol><li>制备100nM的fMLP的和1微克/毫升的Alexa 594的在RPMI 1640新鲜混合物饥饿培养基作为刺激。 注释:fMLP的和Alexa 594的需求混合物被新鲜的，以确保最大的功效。 </li><li>安装一个4孔腔式播种了细胞在一个40X油镜。 </li><li>将荧光显微镜中的多通道配置如下: <ol><li>设置GFP标记之PKD1，红色发射信道（580-620纳米）对趋化fMLP的（Ale​​xa的594），用于识别粘附HL60细胞绿色发射信道（500-530纳米），和透射光信道。 </li><li>开始"活"的采集和优化三个通道的采集条件: </li><li>强度和激光功率对于所有三个通道之间微调，以减少光漂白用于成像的一个较长时期。 </li><li>如果需要的话，平均每2至4个帧以减少背景噪声比更好质量的图像。使用1秒的时间间隔。 </li></ol></li><li>搜索秉承，在GFP和DIC（微分干涉对比）信道的观点表示强烈PKD1-GFP的细胞。坚持细胞在形态平整且没有快速移动。 </li><li>鉴定粘附的HL60细胞高表达的GFP-PKD1，通过降低激光功率优化取得条件，同时提高每个通道的检测器增益，以减少对长期成像（ 图3A）的光漂白后。 </li><li>占用100微升fMLP的/ Alexa的594解决方案可以与200微升移液器，待用。选择"延时收购"，并设置间隔2秒和帧的数量，以获得100个。 注:间隔和框架收购的数量取决于实验的目的。 1-2秒的间隔和100帧通常用于成像实验中， 如图3，对于缓慢迁移细胞，较长的时间间隔是必要的长期成像。相反，对于迅速迁移细胞，需要用于观察细胞迁移的连续细节更短的间隔。 </li><li>使腔室的位置不改变小心脱下4-井室的盖子。立即开始采集并获得未刺激细胞的3-5图像，同时直接在用激光作为指导的光斑的细胞定位200μl的吸移管。 </li><li>移液器被成像具有快速的fMLP的/ Alexa的594混合物中的细胞，甚至流，完成全套时间推移的收购。命名和保存将被进行进一步的数据分析的数据。 </li></OL&gt; 6.成像Chemotaxing细胞的查看和控制化学引诱刺激<ol><li>使用10微升micropipetter和注射技巧，回填微量新鲜配制的fMLP的和Alexa594混合30微升。 </li><li>附加微量到安装在显微镜载物台顶微量保持器和管连接到压力供应单元，提供了一个恒定的压力，从微量释放fMLP的/ Alexa594的溶液，以产生一个稳定的梯度的装置。 </li><li>打开压力供应和设定补偿的压力（PC）至70百帕。 </li><li>安装有超过上共聚焦显微镜或等值荧光显微镜40X油镜表达PKD1-GFP HL60细胞接种1井的腔室盖玻片。 </li><li>坐落在通道模式收购了以下配置:对GFP标记PKD1绿色通道（500-530纳米）;对于趋化fMLP的（Ale​​xa594）红色通道（580-620纳米）;和反mitted光通道识别秉承HL60细胞。 </li><li>启动"活"采集和优化的三个通道的采集条件:调整各信道用于成像数据的定量分析的强度;强度和激光功率之间微调对于所有三个通道，以获得具有最佳质量的图像和最小化光致漂白剂。如果需要的话，平均每2〜4帧更好的影像画质。我们通常使用一个1秒的时间间隔。 </li><li>开始"活"的采集和搜索坚持，在GFP的观点和透射光渠道表示强烈PKD1-GFP的细胞。以可视化的梯度fMLP的用明视场光学中心的视图（ 图4A）的中心场微量和表达PKD1-GFP附着的HL60细胞。 </li><li>选择时间推移采集并设置间隔为2秒的间隔和帧的数量，以获得100个。 注:收集各t之后IME推移系列，名和保存将被进行进一步的数据分析的数据。 </li></ol>

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The authors declare no competing financial interest for this work.

在本研究中，我们表明趋化实验的两个例子:首先，由小区迁移分析装置的多个趋化实验的同时监测;第二，趋化梯度的可视化和信令在实时同一细胞事件的时空动力学。 多个同时趋化实验小区的流动性分析装置在本研究中，我们引入了一个详细的协议来执行使用细胞移动分析装置同时进行多个趋化实验。该设备允许用户观察与传统明场观察的10倍物镜细胞趋化行为。因为流体流的扩散显性特性的，细胞迁移分析装置产生高度可预测的，稳定的梯度，并允许多达六个趋化实验可以同时进行。该协议的关键步骤获得可靠的梯度和对齐单元格的露台线路。用户应严格按照制造商的趋化因子和细胞的固定组件与注入指令。详细说明也可在网上。相比其它趋化方法11-13,15，该装置显著改善趋化实验的可靠性和效率。在尺寸四种尺寸的细胞迁移分析装置的芯片的4，5，6，和8微米的，可容纳不同类型和细胞的尺寸。我们发现，一个4或5微米的细胞迁移分析装置的芯片是适合的HL60和D菌细胞，这是在直径约10-15微米。然而，一个限制是，这个装置不适合于所有类型的细胞。我们不得不使用与Raw267.4细胞中的细胞运动分析装置趋化性试验中收效甚微。其原因可能是Raw267.4细胞迁移速度太慢。高效枝所需要的时间Raw267.4细胞motaxis可能比的梯度由设备维护的时间长得多。相反，转孔迁移实验工作的很好Raw267.4细胞9。另一个限制是荧光观察是不可能与当前设备。未来的方向是监测荧光成像具有较高的放大倍率。这是可能的改进的细胞迁移分析装置，其配备有荧光检测和100X物镜。此外，同步检测次数也提高到12所有这些改进促进趋化实验的增强吞吐量和亚细胞动力学中迁移细胞的观察。 HL60细胞的转染效率高，表达荧光蛋白标记的蛋白质 HL60细胞是一个活跃分裂细胞白血病细胞系，并在悬浮液中生长。据此前报道18-20，HL60细胞至g耐烯转移。既脂质和电穿孔基因转移已经过测试，并与电穿孔，获得更高的转染效率，如在第4节中详细描述的获得高生存能力电穿孔后为由于从电穿孔产生的严重损害获得高转染效率的关键。随后，彻底和温和的细胞处理是必要的，尤其是电后。所有媒体都必须预热并轻轻加入到细胞中电穿孔后的任何步骤。它也是至关重要的细胞的电穿孔试剂的暴露时间最小化。以避免细胞死亡，在电穿孔后，RPMI 1640电恢复介质必须立即向细胞中加入。我们发现，在回收培养基20％胎牛血清，使其比10％FBS的高得多的细胞回收率。电后，在孵化RPMI 1640电恢复中30分钟，是更大的活力和转染的关键效率。为了进一步增加转染效率，我们也使用每转4微克质粒DNA，这是由制造商推荐的质粒的几乎两倍量。 上有电穿孔转染两个主要限制:蛋白表达的顷刻和细胞数的限制（每个转染2×10 6个细胞）。在未分化的细胞，表达仅在第一耦合的细胞分裂是可检测的，因为载体质粒是通过半每次细胞分裂后稀释。为分化的HL60细胞存活不超过48小时。其结果是，任何实验需要新鲜转染实验前6小时。对于一个电穿孔的细胞数仅仅满足为任何生物化学测定的最低要求。对于重复使用或大量的目的，强烈建议在未分化的HL60细胞系建立稳定表达的兴趣WHI的蛋白质通道已经被病毒载体具有标记的荧光蛋白质中，如果病毒载体是可用的，或可以构造。

真核细胞中感测和朝向趋化梯度内浓度较高移动，蜂窝式过程被称为趋化。趋化起着许多生理过程的关键角色，例如胚胎发生1，神经元图案2，癌细胞3的转移，嗜中性粒细胞向炎症4的部位的募集，并且模型生物体粘菌 5的发展。在一般情况下，真核细胞检测用G蛋白偶联受体5的化学引诱物。化学引诱物与这些受体的接合促进了异源的G蛋白Gα和Gβγ，进而活化下游信号转导途径，最终调节肌动蛋白细胞骨架的时空组织以驱动细胞迁移5-9的离解。 细胞生物学家一直致力于开发和趋化改进是测定以检查G蛋白偶联受体（GPCR）介导的信号如何定向细胞迁移。 Boyden小室或透孔迁移实验是由10博伊登于1960年开发的。该测定法的工作原理是创建趋化化合物的由微孔膜分开的两个井之间的梯度。其简单和易用性，使其成为使用最广泛的趋化性试验日期。然而，该测定不能使细胞的迁移过程中被可视化。该Zigmond室是第一款可视微流体装置，可用于在朝着源趋化11穿越狭窄的收缩盖玻片细胞迁移的清晰成像。邓恩12和英索尔13改性，提高了高分辨率和长期成像能力的Zigmond室趋化性试验的。因为流体流动的高度可预测，扩散显性特性的，微流体已经提供下一generati解关于趋化实验如EZ-TAXIScan（小区迁移率分析装置）。 与保证了梯度的稳定，设备允许6趋化实验，以进行同时（ 图1A）。相反在上述各个室测定法产生的定向固定梯度，由冈瑟Gerisch开发的针或微量测定法产生具有可动源14的梯度。在测定中，趋化是从可动微量释放，以产生一个稳定的梯度。有了这个针法，研究人员发现，不同细胞生成具有根本的不同特点伪足。应用荧光显微镜，我们能够以可视化的梯度，以方便在整个15的定量测量。在这项研究中，我们描述了准备HL60趋化（人早幼粒白血病细胞），具体方法同时监控多个趋ASSAYS与细胞迁移分析装置，和可视化信号分子的GPCR介导的时空动力学如蛋白激酶D1在单个活细胞中响应于可见光，spatiotemporally可控趋化刺激。我们的先进的成像方法能够应用于一般的趋化研究，并且特别适用于哺乳动物细胞系统。

<table border="0" cellpadding="0" cellspacing="0" width="459">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">RPMI 1640 Medium GlutaMAX</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:120px;">61870-036</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Sodium pyruvate</td>
			<td style="width:107px;">Thermo Fisher Scietific</td>
			<td style="width:120px;">11360-070</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Fetal bovine serum</td>
			<td style="width:107px;">Gemini Bio-Products</td>
			<td style="width:120px;">100-106</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">1M HEPES sterile solution, pH7.3</td>
			<td style="width:107px;">Quality Biological Inc.</td>
			<td style="width:120px;">A611-J848-06</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Penicillin streptomycin solution</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td align="right" style="width:120px;">15140122</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:120px;">NucleofectorTM 2b</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:120px;">AAB-1001</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="217">
			<td height="217" style="height:217px;width:120px;">AmaxaTM Cell Line NucleofectorTM Kit V including NucleofectorTM Solution, Singe use pipettes, AmaxaTM certified 100 ml aluminum electrode cuvettes</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:120px;">VCA-1003</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">Lab-Tek chambered #1.0 Borosilicate Coverglass</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155383</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">2 % Gelatin solution</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:120px;">G1393</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Fibronectin</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:120px;">F1141</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">HBSS (Hanks&#8217; Balanced Salt Solution)</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:120px;">14025-076</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="39">
			<td height="60" rowspan="2" style="height:60px;width:120px;">Bovine serum albumin</td>
			<td rowspan="2" style="width:107px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:120px;">A3803</td>
			<td rowspan="2" style="width:113px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Single well Lab-Tek II coverglass chambers</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155361</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Four-well Lab-Tek II coverglass chambers</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155383</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Alexa 594</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:120px;">A-10438</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">fMLP</td>
			<td style="width:107px;">Sigma -Aldrich</td>
			<td style="width:120px;">F3506-5MG</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Cover glass thickness 2 22 x 22 mm</td>
			<td style="width:107px;">Corning</td>
			<td style="width:120px;">2855-22</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">EZ-TAXIScan</td>
			<td style="width:107px;">Effector Cell Institute, Inc.</td>
			<td style="width:120px;">MIC-1001</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">EZ-TAXIScan chip (5 mm)</td>
			<td style="width:107px;">Effector Cell Institute, Inc.</td>
			<td style="width:120px;">EZT-F01-5</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">1701RN 10ul syringe</td>
			<td style="width:107px;">Hamilton</td>
			<td align="right" style="width:120px;">80030</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Femtotips II Injection tips</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">5242956003</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Femtotips II</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">930000043</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:120px;">TransferMan NK2, including motor module, X head with angle adjuster, and Positioning aids.&#160;</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">5188900056</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">DIAS software</td>
			<td style="width:107px;">Solltech Inc.</td>
			<td style="width:120px;"></td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="181">
			<td height="181" style="height:181px;width:120px;">LSM 780 META or equivalent confocal microscope with a 40X 1.3 NA or 60X 1.4 NA oil DIC Plan-Neofluar objective lens</td>
			<td style="width:107px;">Carl Zeiss</td>
			<td style="width:120px;"></td>
			<td style="width:113px;"></td>
		</tr>
	</tbody>
</table>

imaging g protein-coupled receptor-mediated chemotaxis and its signaling events in neutrophil-like hl60 cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

利用细胞的流动性分析装置多HL60细胞的趋化的同时成像基于微流体16的原理，制造商已提供的模拟轮廓梯度:梯度在1分钟内产生，在5分钟内稳定，并且保持了2小时。通过微流体所产生的稳定的梯度的高度可预测的轮廓允许多个趋化实验可以同时进行。在本研究中，我们观察到三同时趋化实验（ 图2A和电影1）。我们发现，HL60细胞开始立即chemotaxing的趋化注入趋化的井之后，并保持在直线路径chemotaxing为以下60分钟，与模拟结果为梯度稳定性是一致的。跟踪行进路径和形态细胞的允许定量测量和使用包括总路径长度，速度，方向性，和圆度的细胞（ 图2B）的趋化指数的趋化行为的后续比较。总路径长度为连接路径的质心的线段的长度的总和。速度是通过时间分割的总路径长度获得。方向性向上测量，并且被定义为:（路径的端部的Y坐标减去开头的Y坐标）由总路径长度划分。这给了1.0的对象直接向上移动。小区的圆度的周界的给定的量如何有效地包围面积的度量（以百分数表示）。的圆具有用于任何给定的周长面积最大，并具有100％的圆度参数。直线封闭任何区域，拥有0％的圆度参数。我们显示通过所选择的趋化参数所描述的定量测定的趋化行为（ 图2C）。 一个spatiotemporally查看和控制fMLP的刺激下，在监测HL60细胞PKD亚细胞定位这是一个伟大的技术提前申请荧光标记的和可控的趋化因子刺激的实验系统。在历史上，我们已应用或均质的（也称为统一）刺激或梯度刺激观察细胞响应和行为。然而，"盲"的刺激，不仅提供了关于如何刺激达到细胞无时空信息，也使人们对细胞对刺激的任何"异常"的观察疑问，只是因为我们没有看到刺激。我们以前曾表明，荧光染料（Alexa594）可与趋化被应用于建立趋化浓度以及m之间的线性关系onitored荧光染料强度的15。用绿色荧光蛋白（GFP），荧光染料（Alexa594），并发射的光的红色发射的获取配置，我们能够监视粘附细胞，刺激的应用，并且在细胞响应于刺激（ 图图3A）。蛋白激酶D是说起到引导细胞迁移9,17重要作用丝氨酸/苏氨酸激酶家族。响应于均匀施加fMLP的（红色）刺激，HL60细胞介导GFP标记的蛋白激酶D1（绿色）的健壮膜易位（ 图3B和电影2）。在一个fMLP的梯度（红色）（ 图4A），HL60细胞积极吸纳PKD1前缘（ 图4B和电影3）。的GFP在前缘的突出的亚细胞定位的接近比较表明，PKD1本地化在前缘的后部（图4C）。 <img alt="图1" src="/files/ftp_upload/54511/54511fig1.jpg" /> 图1.细胞迁移分析装置允许多达6个同时的趋化实验。（A）中方案示出了细胞迁移分析装置的芯片的为6独立趋化实验同时监测设计。红显示趋化加入到孔中。 （B）HL60细胞对细胞的井言而趋化扩散，建立一个稳定的fMLP的渐变。 <a href="http://ecsource.jove.com/files/ftp_upload/54511/54511fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54511/54511fig2.jpg" /> 图2.同步监测与HL60细胞多趋化实验的。（A </stro纳克&gt;）蒙太奇显示细胞迁移分析装置趋化性试验的图像来检查上趋PKD特异性抑制剂在0时间和梯度施用后12分钟的抑制效果。趋化HL60细胞用PKD抑制剂1μM的CID755673预处理30分钟。 HL60细胞有或无PKD抑制剂的治疗被允许在任何RPMI1640饥饿中等或100纳米fMLP的渐变12分钟趋化。 （B）的流程示出了跟踪HL60细胞的行进路径长度和形态。趋化作为总路径长度，速度，方向性，和圆度（C）的定量。平均值±SD表示; N = 10，12，或11没有梯度，梯度fMLP的无CID755673治疗，而fMLP的治疗CID755673治疗，分别。 <a href="http://ecsource.jove.com/files/ftp_upload/54511/54511fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3. GPCR介导的PKD1的健壮膜易位响应于均匀施加fMLP的刺激（A）PKD1-GFP（绿色）的多通道监测，趋化（1μMfMLP的0.1微克混合/ ml的荧光染料的Alexa 594，红色） ，和DIC（微分干涉对比度），以确定涂有在RPMI 1640培养基0.2％明胶一个4well室的良好的粘附HL60细胞。比例尺= 10微米。 （B）蒙太奇显示，统一适用fMLP的（红色）诱导PKD1-GFP（绿色）的强劲膜转。 <a href="http://ecsource.jove.com/files/ftp_upload/54511/54511fig3large.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/54511/54511fig4.jpg" /> FIGUR在chemotaxing HL60细胞PKD1电子4.前缘本地化。（A）通道模式收购构造有助于fMLP的渐变的可视化和PKD1的时空动态。在A - C，HL60细胞瞬时表达GFP标记PKD1;可视化从微量（DIC），100nM的fMLP的（红色）产生的fMLP的梯度用0.1微克/毫升的荧光染料的Alexa 594（B）中在chemotaxing细胞的前缘PKD1的富集本地化混合。比例尺= 10微米。 （C）的合并的图像显示，PKD1本地化在HL60细胞的前缘的后面。绿色表示PKD1细胞定位和DIC图像显示前缘的突出区域。比例尺= 5微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54511/54511fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells at JoVE.com

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

视觉趋化实验是为了更好地了解如何真核细胞趋化控制介导的定向细胞迁移是必不可少的。在这里，我们描述了详细的方法:1）实时，多趋化实验的高分辨率监测，和2）同时可视化趋化梯度和信号中的中性粒细胞样HL60细胞事件的时空动力学。

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

imaging-g-protein-coupled-receptor-mediated-chemotaxis-its-signaling

Research

JoVE Journal

Biology

10.0K 次观看.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 视觉趋化实验是为了更好地了解如何真核细胞趋化控制介导的定向细胞迁移是必不可少的。在这里，我们描述了详细的方法:1）实时，多趋化实验的高分辨率监测，和2）同时可视化趋化梯度和信号中的中性粒细胞样HL60细胞事件的时空动力学。 

视频: Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

科研

生物学

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

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