RUM是由美国心脏协会前博士生奖学金（14PRE18560010）的资助。研究是由美国心脏协会的科学家开发格兰特（13SDG14270009）美国国立卫生研究院（1DP2 CA186752-01与1-R01-GM-105646-01-A1），以资助SS

1.准备DNA <ol><li>设计的传感器构造采用模块化克隆方案。请参考β-AR传感器设计1先前详述。 </li><li>按照商业miniprep试剂协议准备DNA和洗脱在2毫米的Tris-HCl溶液，pH值8，在浓度≥750纳克/微升，A 260 / 1.7 280 - 1.9，A 260 / 2.0的230 - 2.29。 </li></ol> 2.细胞培养制备<ol><li>培养的HEK-293T-的Flp-n的细胞在DMEM含有4.5g / L的D-葡萄糖，补充有10％FBS（热灭活的）（体积/体积），1％L-谷氨酰胺补充，20毫米的HEPES，pH值7.5，在37在5％的CO 2°下在潮湿气氛。处理生物安全罩的后续步骤细胞。 </li><li>允许传代于6孔盘前的细胞生长至汇合单层 。时间，以达到融合依赖于初始接种密度。使用板指来到内1汇合 - 电镀2天六个孔电镀。汇合10厘米组织培养处理过的培养皿具有大约4×10 6个细胞/ ml的细胞密度。参见图1为细胞培养生长的图像。 </li><li>洗涤细胞10毫升PBS，并用0.25％的胰蛋白酶trypsinize（见讨论，第2款）。板8×10 5个细胞/孔在组织的2ml培养基培养处理的六孔菜肴，并允许粘附16 - 20小时。 </li></ol> 3.转染条件<ol><li>错开转染对于可能需要不同的时间量，以达到最佳表达构建体（在20 - 36小时）。同步进行统一的实验时间的条件。也有可用于分析过程中的背景噪声和散射减法未转染的对照孔在相当于细胞密度。 </li><li>把转染试剂室温:降低血清培养基，DNA转染试剂。 </li><li>在一个生物安全罩以下列顺序结合试剂在无菌的离心管:混合料2用100μl降低血清介质微克的DNA。穗6微升转染试剂到媒体/ DNA混合物的不接触混合物的表面或管子的一侧。设置各井一转反应。转染条件可以优化（1 - 4的DNA微克，3 - 6微升转染试剂），以取得一致的表达水平。 见表1更加优化的比率。 </li><li> 30分钟 - 在生物安全罩15孵育在室温混合。如果离开孵化时间超过30分钟，不要使用反应。 </li><li>添加到在一个逐滴方式的细胞反应跨孔和轻轻摇动六孔以确保彻底混合。添加好每一个反应。 </li><li>表达20小时后，荧光显示器，利用组织培养荧光显微镜。评估与40X在细胞10X物镜和蛋白质定位种群表达。 OBSERVE在质膜（PM）的蛋白的表达。如大量内部注意到，直到显著表达式在PM检测监控转染。 </li></ol> 4试剂和设备的准备<ol><li>在-80℃的准备100毫米制药股和存储:重酒石酸异丙肾上腺素（100毫米含有300毫米抗坏血酸卫生署2 O）。制作冰/在寒冷的房间，并立即闪冻结。等分试样可以制造和使用长达一年。 </li><li>准备电池溶液（〜2毫升/条件），并存储在37℃水浴。要让每一天都有新鲜。 Cell的缓冲成分参考表2。 </li><li>制备药物缓冲液（10ml）中并在室温下储存。药品成分的缓冲参考表2。 </li><li>酸洗用浓盐酸试管。用弱碱（1μMKOH）中和，并与卫生署2 O彻底冲洗比色皿</li><li>准备工作车站周边的荧光有几个10，200，1000微升枪头，一个计时器10分钟倒计时，有技巧比色皿清洗，棘手的任务湿巾可访问的真空管路，并用超H 2 O.喷瓶箱设置</li><li>热量荧光及热块到37℃外部水浴。 </li><li>打开荧光;设置荧光采集程序收集CFP与激发430nm处，带通8处;发射范围450纳米 - 600纳米，带通4纳米。对于YFP集合只作为传感器控制（见讨论 ）一套激发至490纳米，带通8纳米，发射范围500 - 600纳米，带通4纳米。 CFP收集设置将用于收购在这个实验中一个FRET谱。 </li><li>放置在加热块12的1.5 ml离心管，如下面的图2。这些管子是细胞等分管座（500微升离心管。）将一小块组织的持有者1和7，以减轻放在这里的比色皿。 注意:使用单独的试管未经处理的条件和药物条件防止交叉污染。 </li></ol><img alt="图2" src="/files/ftp_upload/54696/54696fig2.jpg" /> 图2. 微量离心管设置，并在加热块试管位置参考未处理的样品是在位置1。细胞等分管是在位置2 - 6比色皿药物处理过的样品在位置7;细胞等分管是在位置8 - 12。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="9"><li>填充支架2 - 6和8 - 12〜750微升的水以创建微型37℃水浴中。 </li><li>将10 500细胞等分微升离心管到小型水浴场（持有2 - 6，8 - 12）。每个管将是条件的单个重复（5 untreateD，5药物治疗的）。 </li><li>监测表达细胞（见步骤3.6）。 </li></ol> 5.实验和数据收集<img alt="图3" src="/files/ftp_upload/54696/54696fig3.jpg" /> 图3.实验原理图，详细的逐步指导实验设置和执行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>参考图3实验原理图。当细胞准备收获的基础上，用荧光显微镜检测蛋白的表达（见讨论，第4段）:生物安全罩，轻轻取出约1毫升媒体，悬浮细胞在他们的文化与P1,000和悬浮转移入1.5ml微量离心管中。 注意:避免使用胰蛋白酶，因为它可能消化的N端和/或结合GPCR传感器的口袋里。 </li><li>计数细胞，以确保再悬浮适当的细胞密度。优化4×10 6个细胞/ ml重悬浮体积。 </li><li>旋在摆动斗离心机细胞在室温下，290×g离心3分钟。离心后去除上清液。 </li><li> 轻轻重悬细胞于1ml细胞缓冲液（贮存在37℃），并重复步骤5.3。在第二次旋转，从-80收集100毫米毒品股票等分℃。制作1:100稀释药品缓冲1毫米的工作股票，并保持在室温。 </li><li>在第二次离心后，除去上清，轻轻悬浮细胞于1ml细胞缓冲液（4×10 6个细胞/ ml）的。在用1ml细胞和1ml细胞缓冲液作为空白的分光光度计样品的测量的OD 600。分装细胞在一次性塑料杯中，并回传给离心管立即分光光度法以下。 </li><li>对于未转染控制CELL设备状态谱，轻轻悬浮未转染细胞在1毫升细胞缓冲器，P1,000吸管，加入90微升细胞的比色皿，并获得在激发波长430 FRET谱，带通8纳米，发射450 - 600纳米，带通4纳米。收集3 - 5新鲜的90微升细胞重复光谱。保持细胞的库存，在37℃的规格之间，P1,000每个样品等分试样之间轻轻悬浮，并冲洗比色皿用超纯的 H 2ö样本之间。 </li><li>对于实验条件，等分试样90微升转染的细胞以每个固定器2的500微升管 - 6,8 - 在热块12。轻轻悬浮细胞的股票，每个等分之间P1,000吸管。 </li><li>细胞分装后，加入10微升药物缓冲区的管2 - 6未经处理的条件样本。 </li><li>开始加入10微升1mM的药液进入管8的实验中，启动定时器，从10分钟倒数，并轻轻地用P200滴管混合管。关闭管和返回到37°加热块。 </li><li>马上拿起试管2，用P200轻轻混匀（使用新尖），加入90微升细胞悬液，以未经处理的条件比色皿，并在荧光到位。 </li><li>收购在激发波长430，带通8纳米，发射450 FRET谱 - 600纳米，带通4纳米。 </li><li>在9分 - 10秒，秒杀管9与10微升1 mM的药物溶液，轻轻地用P200（使用新尖）混合，并返回管散热模块。 </li><li>重复步骤5.10 - 5.11管3和5.12与管10。 </li><li>以1分钟的间隔（08:10，07:10， 等等 ），直到光谱收集所有未处理的条件的样品，及药物已被添加到所有药物条件样品5.13 -重复步骤5.10。选用一个新鲜提示为每个吸管步骤防止交叉污染。 </li><li>在5分钟 - 10秒，开始搅拌筒8（药物条件）轻轻地用吸管P200，加入90微升细胞悬液分离试管中荧光药物处理的样品和地点。 </li><li>收购˚FRET谱（见步骤5.11设置）。 </li><li>以1分钟的间隔（04:10，03:10， 等等 ），剩余药物样本条件（管9 - 12）5.16 -重复步骤5.15。 </li><li>实验结束后，保存项目文件，彻底清洗试管用超H 2 O和重组股管的一个条件。小心防止在洗涤步骤的交叉污染。改变对H 2 O瓶以及在洗涤之间的真空管线的尖端。 </li></ol> 6.数据分析<ol><li>保存并在SPC导出的数据文件格式，以用于分析。分析程序可以从Sivaramakrishnan实验室发布的网站上下载。 </li><li>创建分析软件路径的文件，其中包括分析程序（V9，V15），未转染的样本文件（见步骤5.6未转染的细胞收集频谱），输出数据文件，逗号分隔值（CSV）数据输入文件。 </li><li>输入以下信息成CSV文件（见表3样品），并指定各条件对于每个样品，其中包括: 文件名 - 个人SPC图形文件 受体-候其中GPCR构建体进行了测试（ 例如，Β2） 粘合剂-候该构建体的肽变体进行了测试（ 例如 ，S） 激动剂 - 候未处理（N）或药物处理（D）的条件 目录 - 在SPC文件保存，通常是由举办日期的文件夹路径 OD - 从分光光度计样品的记录光密度</li><li>输入未转染的样品（步骤5.6）的文件名来减去样本缓冲区和散射噪声。 </li><li>进入条件进入分析程序。 </li><li>运行程序，分析单独的条件（V9）内和跨环境（V15）的样品。 </li><li>排除这是在数据集中离群明显的示例文件，或者通过增加或减少INDIVI OD值调整减法双文件。 </li><li>数据导出到输出文件访问计算FRET比（525纳米/ 475纳米）。 </li><li>通过从个人的FRET比率减去未处理状态的平均FRET比率处理（药物）的条件下计算ΔFRET。 </li></ol>

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作者宣称他们没有利益冲突。

在这个系统中FRET测量的严密动态范围强化敏感的质量控制在本协议中的每一个步骤的必要性。以确保成功的FRET实验的最重要的步骤是:1）细胞培养，2）转染3）蛋白表达和4）及时，准确协调的测定执行期间。 细胞的健康和维护/电镀质量可以对实验系统和差细胞健康的信号 - 噪声一个显著影响可以使它不可能检测在FRET任何一致的变化。保守，细胞是健康的大约20代，虽然这可能会因细胞系，处理和培养条件。一旦细胞有困难成长为一个汇合单层，或者开始在更多的树突图案（参见图1（b）），实验性的背景噪声会受到不利影响持续增长。仔细细胞的维护，包括日常MEDI一个变化，并定期从维护板除去未贴壁细胞和碎片，将增强细胞的质量为六孔电镀和转染。细胞团块，其中转染效率产生不利的影响，可以有效地分离成单个细胞通过胰蛋白酶消化的维护板:治疗10厘米汇合菜肴用10ml 0.25％胰蛋白酶，30秒，除去胰蛋白酶但留下约200微升，代替盘在37 °C培养箱为2 - 3分钟。细胞会很容易脱落菜，都结块不敏感。 关键是要优化该实验中的转染步骤。六井必须理想地60 - 高效转染和最佳表达80％汇合。如果太少细胞已粘附（&lt;60％），等待约2 - 6小时转染，或直到细胞的至少70％的附着。六阱中超过汇合（&gt; 80％）也将减少的转染效率。转染在较低细胞汇合增加细胞死亡率。 DNA的浓度和纯度也很关键（见步骤1.2）。使用低浓度和/或质量差的DNA制备产生负面影响转染效率转染条件可以为每个结构进行调整，请参阅表1以获取更多信息。 利用组织培养荧光显微镜蛋白表达的准确和一致的监测是在这个过程中又一个关键步骤。虽然这一步骤是受个人的判断，也可以使用其他技术，例如显微术，并监控随时间的表达定量，虽然它们没有在这里详细描述。因为我们已经在我们的系统测试成功构建，表达大约需要18-36小时才能达到最佳的表达。根据我们的经验，构建了这个时间窗口期间显示不佳表现后40小时很少改善。将构建我们与没有表现出degradatio的迹象出版N，但是这可能是某些GPCR的一个问题。在我们的试验中，传感器降解有可能在转染时间超过30小时。传感器完整性使用YFP / CFP比例进行测试:525纳米从YFP激发（490 nm）的光谱阅读，和475纳米的距离CFP激发（430 nm）的光谱阅读。对于设置，请参阅步骤4.6。推荐的YFP / CFP比率在1.7的范围内 - 2.0，以1.8的理想比例。这个值取决于相对于CFP14 YFP的近似两倍更大的亮度。因此以最小的降解的组成传感器将包含荧光团和具有YFP:约2 CFP:1的比例。传感器质量已被证实后，以确认蛋白定位和表达在细胞膜是重要的。显著胞内表达可以是蛋白质的降解，内化，或蛋白质的持续贩卖的结果。显示器构造随着时间的推移，以查看是否表达在细胞膜增强。接下来的暴击在表达iCal的挑战是转染效率。约70％+转染效率是必要的适当的信号 - 噪声检测在该荧光计系统。如果较少的细胞被转染，信号对噪声的量可能仍然是可检测的，但将在一个FRET实验样品间要少得多一致。这将系统的窄动态范围内妨碍准确的数据分析。表达水平也呈现显著障碍在实验过程中实现充足的信号 - 噪声。对于可检测的表达水平通过荧光计，1.5 475纳米和450纳米（细胞散射）之间的信号的比率是足够用于检测FRET发生了变化，但是最佳表达将具有约2.0+的比率。作为参考，β2-AR数据在信号与噪声范围的4×10 5 CPS（450 nm）到1×10 6 CPS（475纳米），信噪比2.5收集。这个比例将有助于减少variabi量样本，也可能会影响数据分析之间lity。这些表达水平也受到灵敏度和荧光光学的最佳比对;其它系统可能需要足够的信号 - 噪声优化不同的参数。 细胞对时间和温度非常敏感。一旦实验过程已经开始，柔和处理是必不可少的，以避免细胞死亡。可采取的具体措施，后勤加快这一进程，并避免错误及时准备，包括在工作站，确保所有设备工作正常，事先并规划出实验的目标。理想的是收获的30分钟内使用的细胞，并在我们的系统中，该协议是在20分钟内执行。一旦技术被掌握，特别染优化和锻炼本身的手巧，该实验可用于各种构建相互比较，生成剂量 - 重sponse曲线，并且传感器可扩展到全长G蛋白。 虽然这里使用的FRET传感器处于GPCR FRET传感器独特的发展，这种特定的实验装置中详述作为用于传感器的实施充分表征测定。基于荧光的测定法使细胞的大量人口在每个实验进行评估，并且不依赖于纯化的蛋白质或膜用制剂，因此维持体内环境。这个实验设计也得到了优化，以检测于GPCR的激动剂刺激系统中看到非常小的变化。

G蛋白偶联受体（GPCR）是七跨膜受体。人类基因组中单独含有约800个基因编码的GPCR，这是通过各种配体包括光，添味剂，激素，肽，药物和其它的小分子激活。目前市场上的目标GPCR的所有药品的近30％，因为他们在许多疾病状态2发挥很大的作用。尽管几十年来对这个受体家族丰富的工作经验，有留在场上显著悬而未决的问题，特别是关于推动GPCR-效应相互作用的分子机制。迄今为止，只有一个高分辨率晶体结构已发表 ​​，提供洞察β2 -肾上腺素能受体（β2 -AR）和G蛋白3之间的相互作用。在过去的三十年的广泛研究一起，它重申一个具体结构部件即临界在此互动:Gα亚基C端。这种结构是两个G蛋白活化的GPCR 4和G蛋白选5-6重要。因此，GαC端提供了GPCR的配体刺激和选择性激活G蛋白之间的关键环节。 研究在过去十年中表明的GPCR填充广泛的构象的风景，配体结合的稳定的GPCR的构象的子集。虽然若干技术，包括结晶，核磁共振和荧光光谱，和质谱可用来检查GPCR的构象的风景，有办法一个缺乏以阐明在效应选择7的功能意义。这里，我们概述一个Fӧrster共振能量转移（FRET）基的方法来检测G蛋白选择性GPCR的构象。 FRET依赖于两个荧光团的接近和平行取向具有重叠发射（供体）一次激励（受体）谱8。作为供体和受体荧光团来靠得更近如蛋白质或任一构象变化的蛋白质-蛋白质相互作用的结果，它们之间的FRET增加，并且可以使用各种方法8进行测量。基于FRET的生物传感器已经在GPCR领域9广泛应用。他们已被用于在第三细胞内环和GPCR C端插入供体和受体，探讨在G蛋白偶联受体构象变化;传感器被设计用FRET对10分开贴标GPCR和效应（G蛋白亚基/抑制蛋白）来探测GPCR和效应的相互作用;一些传感器也检测所述G蛋白11的构象变化。这些生物传感器已经启用了现场询问，包括在GPCR和构象变化的效应，GPCR-效应相互作用动力学和变构配体12悬而未决的问题，众说纷纭。我们的组在创建一个能检测激动剂驱动的条件下，g的蛋白构象GPCR生物传感器特别感兴趣。这种生物传感器依靠命名为痉挛（系统蛋白亲和强度调制）13最近开发的技术。 SPASM涉及到使用ER / K连接，控制它们的有效浓度圈养相互作用蛋白结构域。侧翼带有FRET对荧光团的链接器创建一个工具，它可以报告蛋白12之间的相互作用的状态。先前1使用了痉挛模块到GαC-末端系绳到GPCR并监控其与FRET的荧光团的相互作用，mCitrine（通过其通常已知的变体，黄色荧光蛋白（YFP），激发/发射峰在提及的这个协议525分之490纳米）和mCerulean，激发/发射峰四百七十五分之四百三十零纳米）（由它的俗称变种青色荧光蛋白（CFP在本协议中提到的）。从N至C末端，T他的遗传编码的单一多肽包含:全长GPCR，FRET受体（mCitrine / YFP），10纳米的ER / K连接体，FRET供体（mCerulean / CFP）和GαC-末端肽。在这项研究中，传感器被简称为GPCR-接头长度-Gα肽。所有部件都通过一个非结构（甘氨酸-丝氨酸-甘氨酸）4连接子，使每个域的自由旋转分离。以前用两个典型的G蛋白偶联受体进行此类传感器的详细特征:β-AR和视蛋白1。 此传感器被瞬时转染到HEK-293T细胞和基于荧光 - 活细胞实验的FRET对中在存在或不存在配位体的每秒（CP​​S）计数的任意单位的测量荧光光谱。这些测量用于计算荧光团（YFP 最大值 / CFP 最大值 ）之间的FRET比率。在FRET（ΔFRET）的变化，然后减去平均FRET比率计算从配体处理的样品的FRET比率的未处理的样品。 ΔFRET可以跨结构进行比较（β-AR-10纳米Gαs肽与β-AR-10纳米无肽）。这里，我们详细协议来表达活的HEK-293T细胞，这些传感器，监视它们的表达，并安装，执行，和基于荧光 - 活细胞的分析FRET测量未处理与药物处理的条件。虽然该协议是专用于与100μM的异丙酒石酸氢盐处理的β2 -AR-10纳米Gαs肽传感器，它可以为不同的GPCR-Gα对和配体进行优化。

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">B2-AR-10 nm- Gas peptide sensor</td>
			<td style="width:144px;">Addgene</td>
			<td style="width:148px;">47438</td>
			<td style="width:547px;">https://www.addgene.org/Sivaraj_Sivaramakrishnan/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:144px;">Fermentas/Fisher Sci</td>
			<td style="width:148px;">FERK0503</td>
			<td>Elute in 2 mM Tris elution buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">HEK-293T-Flp-n cells</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">R78007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Trypsin (0.25%)</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DMEM- high glucose</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">11960-044</td>
			<td>Warm in&#160; 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FBS, certified, Heat inactivated, US origin</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10082147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">Glutamax I 100x</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">HEPES</td>
			<td style="width:144px;">Corning</td>
			<td style="width:148px;">MT25060CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Opti-MEM</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">31985-070</td>
			<td>Reduced serum media; Bring to room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">XtremeGene HP transfection reagenet</td>
			<td style="width:144px;">Roche</td>
			<td style="width:148px;">6366236001</td>
			<td>Highly recommended for its consistency. Bring to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FluoroMax 4</td>
			<td style="width:144px;">Horiba</td>
			<td style="width:148px;"></td>
			<td>Use with FluorEssence V3.8 software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">3-mm path length quartz cuvette</td>
			<td style="width:144px;">Starna</td>
			<td style="width:148px;">NC9729944(16.45F-Q-3/z8.5)</td>
			<td>May require cuvette holder/adaptor for use in Fluorometer, available from Starna</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Sc100-S3 Heated Circulating water bath pump</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:148px;">13-874-826</td>
			<td>Warm to 37 &#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">Thermomixer Heat Block</td>
			<td style="width:144px;">Eppendorf</td>
			<td style="width:148px;">22670000</td>
			<td>Warm to 37 &#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ultrapure DNA/RNAse free water</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10977015</td>
			<td>Use at room temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">D(+)-glucose, anhydrous</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">G5767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">aprotinin from bovine lung</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">leupeptin hemisulfate</td>
			<td style="width:144px;">EMD</td>
			<td style="width:148px;">10-897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">L-ascorbic acid, reagent grade</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(-)-isoproterenol (+)-bitartrate</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">I2760</td>
			<td>Use fresh aliquot each experiment</td>
		</tr>
	</tbody>
</table>

g protein-selective gpcr conformations measured using fret sensors in a live cell suspension fluorometer assay

Fӧrster共振能量转移（FRET）基中的GPCR信号传导的调查研究已变得越来越普遍。我们的研究小组开发出一种分子内FRET传感器来检测Gα亚基及以下激动剂刺激活细胞G蛋白偶联受体之间的相互作用。在这里，我们对细节处理后用100μM盐酸异丙将β2肾上腺素能受体和GαsC端肽的检测FRET变化协议作为以前的特点1。我们FRET传感器是由一个全长的GPCR，一个FRET受体荧光团（mCitrine）的串联的单个多肽，一个ER / K痉挛（系统蛋白亲和力强度调制）连接体，FRET供体荧光团（mCerulean）和GαÇ末端肽。该协议将详细介绍细胞制备，转染条件，设备安装，试验执行和数据分析。这个实验设计检测小通道安格斯在FRET指示蛋白质 - 蛋白质相互作用，并且也可以用来比较相互作用的跨越配体和G蛋白偶联受体，G蛋白配对的强度。增强信号噪声中的测量，该协议要求在所有步骤升高的精度，并且在这里提出，以使重现的执行。

实验的广义示意设置和执行在图3中详细说明。 为了检测在传感器的窄动态范围内的FRET的变化，它是附着在系统的细微差别粘附到临界。小区质量势在必行蛋白质表达以及在抽样保持一致。 图培养细胞以一致的单层（10X），它是最佳生长的1特征的图像为六孔电镀和转染图1（a）和结块图案生长的细胞铅树突形状图未建议一致电镀1（b）中 。转染条件也可以实现可再现的表达进行了优化。几个条件已为这里使用的推荐的转染试剂进行优化。 表1详细这些条件，可降低血清介质。 DNA和转染REA绅士的比率。 一旦所有数据被收集和数据输入到CSV文件进行分析（ 见表3样品），所产生的结果将类似于图4中所示的原始FRET光谱数据（a）和归一化的，平均的FRET光谱如图图4（b）。 图4中的红色光谱是在未处理的样品和蓝色被药物处理的样品。所有在图4（a）该原料FRET光谱有一致的数据分析（ 即 ，CPS在450nm在475nm处相对于CPS）足够的信号对噪声的范围。与该范围内，光谱是平滑和来自样品的水峰（喇曼峰值是在500nm处）也是最小的。表达水平低导致在数据分析带来干扰的突出水峰。数据是在475纳米的归一，设置该值的CPS至1.0（ 图4的（b））。在对β-AR这组数据-10纳米Gαs肽传感器，有在525纳米的未处理的（红色）和处理过的（蓝色）样品之间读取显著变化。该ΔFRET变化是从这些数据集的FRET比率（525毫微米/ 475毫微米）来计算，并且通过输出文件进行访问。 如果蛋白质表达低，有转染效率较差，或者在反应杯为荧光读数低细胞密度，光谱可能会出现更大的声音， 如图5，相对于图4（a）该信号与噪声范围为该数据集是不理想的，在约1.0 - 1.6，与CPS 最大的4×10 5个该低表达水平有助于在原始数据图看到的锯齿状光谱图5（a），然而，数据很紧，能够成为归一如图5（b）。虽然水峰（〜500纳米）不排队完全之间的数据集图5（b）本恩periment仍用于分析可用的。 图6是代表一个实验是不足的分析。而信号-噪声样本的是约2 - 3为处理（蓝色）和未处理的（红色）的样品，在整个样品试管细胞密度太低图6（1×10 5 450纳米的值）（一个）。这会在减去背景和规范化图6（b）和频谱不对齐的问题。减法可以通过增加或表3中减少的OD的样品进行调节。然而，即使具有低细胞密度，水峰（〜500纳米）变得更大，喧闹如图6（c），并且抑制从进一步分析该数据集。 <img alt="图1" src="/files/ftp_upload/54696/54696fig1.jpg" /> 图1.培养细胞生长的实施例。将细胞以单层生长（一）是理想的一致电镀和转染。出现在团块或树突状图案（b）可以不六阱始终如一板生长的细胞，可以显示转染效率差，并且可能会在FRET频谱的振幅创建不一致。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig1large.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/54696/54696fig4.jpg" /> 图4.代表性的数据分析β-AR-10 纳米Gαs多肽药物±。原始数据的形象代表 （a）和归一化的，平均数据（b）与用未经处理的（红色）样品和处理（蓝色）的β2 -AR-10纳米Gαs肽传感器收集的小号后100μM异丙肾上腺素酒石酸5分钟的孵化amples。在475nm处，在525nm处YFP 最大发射CFP 最大发射。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig4large.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/54696/54696fig5.jpg" /> 图5.分析原始和标准化数据不佳蛋白表达的。嘈杂的原始数据谱（a）的表达水平低，每个样品的细胞密度低，和/或构建的转染效率差的结果。这组数据仍然是解释成标准化（B），虽然它并不理想。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6.在荧光计比色皿与原始低细胞密度的分析和归一化数据集。每个样品的细胞密度低，在原始数据（a）可知复杂水/背景减除（B，C），使该数据设置不可解释。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 条件 </td><td> 0.5X </td><td> 0.7倍 </td><td> 1X </td><td> 2倍 </td><td> 2X * </td></tr><tr><td>脱氧核糖核酸</td><td> 1微克</td><td> 1.4微克</td><td> 2微克</td><td> 4微克</td><td> 4微克</tD&gt; </tr><tr><td>降低血清培养基</td><td> 100微升</td><td> 70微升</td><td> 100微升</td><td> 100微升</td><td> 200微升</td></tr><tr><td>转染试剂</td><td> 3微升</td><td> 4.2微升</td><td> 6微升</td><td> 6微升</td><td> 12微升</td></tr><tr><td colspan="6"> *注:推荐异常艰难的结构。一定要小心，因为这种情况导致的高细胞死亡。 </td></tr></tbody></table> 表1.染条件。此表包括试剂的使用推荐的转染试剂转染于2ml井HEK-293T细胞的最佳条件。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"> 细胞缓冲区 </td><td></td></tr><tr><td> 卷 </td><td> 试剂 </td><td> 注释 </td></tr><tr><td> 9毫升</td><td>超纯DNA酶/ RNA酶自由水</td><td></td></tr><tr><td> 1毫升</td><td> HBS（10倍），pH值7.4，储存在4°C: </td><td></td></tr><tr><td></td><td> 200毫米的HEPES </td><td></td></tr><tr><td></td><td> 50毫米氯化钾</td><td></td></tr><tr><td></td><td> 450毫米的NaCl </td><td></td></tr><tr><td></td><td> 20毫米氯化钙2 - H 2Ø </td><td></td></tr><tr><td></td><td> 10毫米氯化镁2 - H 2Ø </td><td></td></tr><tr><td> 100微升</td><td> 20％的D-葡萄糖</td><td></td></tr><tr><td> 15微升</td><td>抑肽酶（ 用蒸馏水O 1毫克/毫升） </td><td>防止降解</td></tr><tr><td> 15微升</td><td>亮肽素（ 用蒸馏水O 1毫克/毫升） </td><td>防止降解</tD&gt; </tr><tr><td> 100微升</td><td>抗坏血酸（100毫米卫生署2 O）* </td><td>稳定激动剂</td></tr><tr><td></td><td> *在开始检测前立即加入</td><td></td></tr><tr><td></td><td></td><td></td></tr><tr><td colspan="2"> 药品缓冲区 </td><td></td></tr><tr><td> 卷 </td><td> 试剂 </td><td> 注释 </td></tr><tr><td> 9毫升</td><td>超纯DNA酶/ RNA酶自由水</td><td></td></tr><tr><td> 1毫升</td><td> HBS（10倍），pH值7.4，储存在4°C: </td><td></td></tr><tr><td></td><td> 200毫米的HEPES </td><td></td></tr><tr><td></td><td> 50毫米氯化钾</td><td></td></tr><tr><td></td><td> 450毫米的NaCl </td><td></td></tr><tr><td></td><td> 20毫米氯化钙2 - H 2Ø </td><td></td></tr><tr><td></td><td> 10毫米氯化镁&lt;子&gt; 2 - H 2Ø </td><td></td></tr><tr><td> 100微升</td><td>抗坏血酸（100毫米卫生署2 O）* </td><td>稳定激动剂</td></tr><tr><td></td><td> *在开始检测前立即加入</td><td></td></tr></tbody></table> 表2. 缓冲成分，该表详细列出用于制作这两种细胞缓冲药品缓冲在实验中使用的试剂。使实验中的每一天都新鲜的缓冲区;存储信元缓存器，在37℃，和药物缓冲器室温。 <img alt="表3" src="/files/ftp_upload/54696/54696tbl3.jpg" /> 为分析表3 CSV文件示例。这个示例数据文件突出了一个实验之后设置的条目。多个实验可以进入同一个CSV文件，如果有必要可以在"加法"列下可以看出端倪。每行必须包含以下信息填写: 文件名​​-个人SPC图形文件受体-指定哪一个GPCR结构进行了测试（ 例如 ，Β2） 粘合剂-候该构建体的肽变体进行了测试（ 例如，S） 激动剂 - 候未处理（N）或药物处理（D）的条件 目录 - 在SPC文件保存，通常是由举办日期的文件夹路径 OD - 分光光度计记录样品的光密度 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54696/54696tbl3large.jpg" target="_blank">请点击此处查看该表的放大版本。</a>

Watch this Scientific Journal Video about G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay at JoVE.com

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

在活细胞悬浮液荧光测定G蛋白选择性GPCR构象测量使用FRET传感器

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

g-protein-selective-gpcr-conformations-measured-using-fret-sensors

Nanyang Technological University

Research

JoVE Journal

Biology

9.5K 次观看.  University of Minnesota. Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

视频: G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

科研

生物学

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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 ...

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 Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

基于细胞健康监测的实时细胞成像分析对低密度脂蛋白胆固醇的吸收检测

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

活细菌中蛋白质-蛋白质相互作用的FLIM-FRET测量。

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

使用活细胞中的构象FRET生物传感器实时监测极光激酶A活化

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...