Name: real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor
Uploaded: 2016-01-04T15:00:01.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

This work was supported by ERC starting grants (ERC-2011-StG 280255 and ERC-2013-StG 337479) and by the Netherlands Organization for Health Research and Development (Vidi 91713319).

1.瞬时转染注:CytoBAS和NucleoBAS（请参阅材料表）被成功地应用于多种细胞类型，（U2OS，将Huh7，HepG2细胞，H69，MDCK和HEK293T细胞）。主要的要求，以使用传感器是，它需要被表达，要求编码DNA进入细胞。 <ol><li>收获细胞从80％汇合为 25cm 2烧瓶中。稀释细胞在完全培养基适用于特定细胞系（10％FBS，1％L-谷氨酰胺，1％青霉素/链霉素的U2OS和Huh7细胞）。 </li><li>板的细胞在所需的密度进入无菌8孔腔室盖玻璃（0.8厘米2）。旨在用于副汇合（60-80％汇合）细胞在实验当天的层，尽管这不是必须的。 </li><li>添加完全培养基，以每孔400微升的最终体积。允许细胞附着24小时。 </li><li>通过加入0.5 NucleoBAS微克制备转染混合物在无菌管或CytoBAS脱氧核糖核酸至100μl培养基（血清/无抗生素）。涡良好。添加转染试剂和涡旋混合。转染试剂，让最佳的效率是细胞类型依赖性，可能需要先测试。 <ol><li>例如，可以使用2.5微升1mg / ml的聚乙烯亚胺（PEI），并培育该DNA / PEI混合15分钟，在RT。 </li></ol></li><li>在液滴细胞添加转染混合物，并用手轻轻晃动孔板混匀。对于〜9.6厘米2井加入100微升的混合物。使用10微升转染混合物为瞬时转染细胞各个成像室的。 注意:在24-48小时，细胞将表达胆汁酸和传感器可用于实验。 </li></ol> 2.稳定转染<ol><li>收获细胞从80％汇合为 25cm 2烧瓶中。稀释沉淀在完全培养基适用于特定细胞系（10％胎牛小号erum，1％L-谷氨酰胺，1％青霉素/链霉素的U2OS和Huh7细胞），以每毫升150,000个细胞的浓度，并在6孔培养板（2毫升端体积）添加每孔大约300,000细胞。 </li><li>允许细胞附着24小时。 </li><li>通过添加0.5 CytoBAS或NucleoBAS的DNA微克准备转染混合物在无菌管（请参阅材料表 ），以100微升培养基（血清/无抗生素）。涡良好。添加转染试剂和涡旋混合。 <ol><li>例如，可以使用2.5微升1mg / ml的聚乙烯亚胺（PEI），并培育该DNA / PEI混合15分钟，在RT。 </li></ol></li><li>加入100微升液滴向细胞转染混合物，并通过用手轻轻晃动孔板混合。建立稳定的细胞系时，总是包括未转染的控制。 </li><li>生长细胞O / N在37℃，5％ 的 CO 2。 </li><li>根据制造商的协议Trypsinize细胞（细胞孵育在37℃瓦特第i个1.5 ml的胰蛋白酶预热每为 25cm 2细胞培养物）和自旋下来，在250×g离心在RT 5分钟。在13ml完全培养基中取出上清，重悬细胞。分裂的细胞在不同直径10厘米的培养皿中:0.5毫升（低密度），1.5毫升（中密度），和4.5毫升（高密度）。执行相同的剩余6.5毫升。 </li><li>加入培养基至10ml的终体积。对于U2OS细胞，用800微克/毫升G418的质粒与新霉素抗性盒，如CytoBAS和NucleoBAS结构来选择阳性细胞。此浓度是细胞类型依赖性的，可以通过选择最低抗生素（G418）的浓度，其中未转染细胞在2至10天（800微克/毫升G418的U2OS细胞）死亡来确定。 </li><li>刷新用适当的抗生素（G418）的培养基中每72小时直到未转染细胞都死了。 </li><li>检查显微镜下培养板20倍的目标为阳性菌落（荧光可以使用与用于绿色，青色或黄色荧光最滤波器集来监测）。 </li><li>标记位于分离自其他菌落与培养皿的外部底部笔的阳性菌落。 </li><li>用磷酸盐缓冲盐水（PBS）中洗涤平板两次，吸它。约有15无菌克隆缸和蘸入无菌有机硅。通过对培养皿轻压应用它们围绕所选的菌落，并确保不超过一个菌落被包括在克隆环。 </li><li>吸取20微升克隆气缸中预热的胰蛋白酶（1倍，0.25％），并在37℃，直到大部分的细胞是否已向上舍入的（观察用显微镜）。 </li><li>添加150微升培养基与克隆气缸血清和合适的抗生素（10％FBS和800微克/毫升G418的U2OS和Huh7细胞）和吸管上下数次以悬浮细胞，并转移到一个96孔板中。 4-24小时后刷新中，使细胞在未来几天内（37℃，5％ 的 CO 2 U2OS和Huh7细胞）生长汇合。 </li><li>刷新介质与抗生素，每3天或4天。当细胞已经逐渐汇合，将它们转移到一个较大的孔板。重复此步骤，直到文化是实验足够大。冻存一些小瓶进行备份。冷冻保存培养基由20％FBS和20％DMSO的培养基（DMEM），而不补充剂。 注意:现在，细胞系是单克隆和认为是稳定的用于表达胆汁酸传感器。抗生素G418（400微克/毫升）的浓度的一半现在足以维持表达在稳定的细胞系。 </li></ol> 3.慢病毒转导注意:某些细胞系被认为是难以通过更传统的方法，如聚乙烯亚胺（PEI）的方法转染。细胞的病毒转导是一种有效的替代工具˚F或基因递送和稳定的转基因表达。 <ol><li>收获细胞从80％汇合为 25cm 2烧瓶中。稀释细胞在完全培养基适用于特定细胞系（10％FBS，1％L-谷氨酰胺，1％青霉素/链霉素的U2OS和Huh7细胞），并每孔在6孔培养板中添加周围200,000个细胞。 </li><li>添加培养基以每孔2毫升最终体积。允许细胞附着24小时。 </li><li>通过培养细胞进行病毒转导4-6小时，用500微升CytoBAS慢病毒的培养基（应要求提供）充满至1毫升完全培养基含有10微克/毫升二乙氨基乙基（DEAE） - 葡聚糖或其他慢病毒总量转导增强。不要忘了包括与未转染对照细胞井。 </li><li>除去培养基，并添加2毫升完全培养基的含有所需抗生素（500微克/毫升潮霉素）。使用慢病毒结构的CytoBAS传导提供小号潮霉素抗性细胞。生长所需的条件下的细胞。 </li><li>刷新培养基（500微克/毫升潮霉素）的抗生素，每3天，分裂细胞时80％汇合，直到所有的阴性对照细胞是死的（对于大多数细胞系大约需要1-2周）。该细胞系目前被认为是稳定表达所述胆汁酸传感器。 <ol><li>或者，使用细胞进行实验病毒转导后1-3天。由于活病毒可能存在，这需要FRET的读出设备，配备了相应的安全级别。 </li></ol></li><li>或者，对于稳定细胞系的快速分离，进行荧光激活细胞分选（FACS）。 <ol><li>收获的细胞从汇合160个厘米2烧瓶中。 </li><li>稀释细胞的Leibovitz培养基（L-15培养基）&lt;5％血清以最大5×10 6每毫升细胞的浓度</li><li>收集排序细胞，补FACS管用1ml收集介质中的（L-15培养基+ 1.5％青霉素/链霉素，1％L-过剩和20％血清）。 </li><li>排序细胞茶晶（520-580纳米）或天蓝色（450-520纳米），兴奋与405激光。 </li><li>排序约500,000个细胞后，减速或停止细胞（5分钟，250×g离心），重悬它们在5毫升正常的完全培养基（10％FBS，250微克/毫升潮霉素为U2OS和的Huh7 CytoBAS细胞）和板他们在一个T25培养烧瓶中。生长所需的条件（37℃，5％CO 2的U2OS和Huh7细胞）下的细胞。 </li></ol></li></ol>胆汁酸传感器4.活细胞成像注:含胆汁酸转运体细胞可以在培养基中去除亲脂性化合物，1-10％的木炭过滤的血清培养。正常血清通常含有胆汁酸，它可能导致细胞内的胆汁酸的胆汁酸传感器的积累和饱和度。 <ol><li>利用共聚焦显微镜FRET测量<ol><li>板细胞稳定或transiently表达该传感器在所希望的密度进入无菌8井盖玻片有底室载玻片（0.8厘米2）。生长的细胞在完全培养基（使用木炭过滤血清）使得在实验当天，汇合是60-70％左右。 </li><li>用200微升1×PBS或Leibovitz的L-15培养基洗一次贴壁细胞在玻片。 </li><li>吸并更换300微升Leibovitz的L-15培养基所以没有二氧化碳控制是必要的。 注:DMEM培养基无酚红，可作为良好，但需要的 CO 2的缓冲条件。胆汁酸的传感器显示（部分），PH值，检测灵敏度，使目标是保持pH值恒定的数据收集过程中。 </li><li>稀释在共焦成像实验，可以在L-15的培养基中使用的化合物。考虑到在成像期间，已经被添加在腔室相对大量的液体来固定样品（50-100微升）的快速混合，所以要3-5x的解决方案。 </li><li>启动共聚焦显微镜的成像软件用37℃的孵化室，并打开紫色405 nm激光上。放一滴浸没油的到目标，并放置在它的上面的8室。对于FRET单细胞成像，使用63X油目标。该传感器已被成功地用于在RT，但细胞的行为可能会被改变。 </li><li>正确设置共聚焦显微镜的设置。 <ol><li>设置采集模式:XYT（单焦平面的时间推移成像）。 </li><li>获得在20秒的时间间隔的图像，以允许采集之间加的化合物无需暂停实验。 </li><li>设置光谱范围内对排放检测:蔚蓝:450-520纳米;黄晶:520-580纳米。 </li></ol></li><li>重点利用透射光的单细胞层上。开始成像和更精确地调节z位置。调整增益和失调的每个通道从背景区分信号的同时，保持荷兰国际集团远低于饱和度的感兴趣的细胞的所有像素。 </li><li>画圈圈来定义感兴趣区域（ROI）的区域。选择的细胞表现出类似的荧光强度。避免细胞明显从平均电池的尺寸和形状不同。暴露细胞的光的时间尽可能短，以尽量减少传感器的光漂白。最好是绘制的ROI不非常接近的细胞周长。此区域是最敏感的，由于焦漂移（细胞移动在z方向）使之更难以监测在实验过程中变化的荧光比率荧光强度的变化。 </li><li>先从共聚焦显微镜测量。等到蔚蓝和黄水晶荧光稳定。 </li><li>测量过程中添加选定的时间点50-100微升胆汁酸或其他化合物。相对高量的液体（五分之一到三分之一的最终体积的）是必要的，以及混合（10秒内）的液体无晃动/对ipetting上下。确保不触及8孔室枪头的边缘，并添加液体慢慢地使细胞不出去焦点。平台期到来之前不要添加新的化合物。这大约需要200秒。 </li><li>结束每个实验用加入了100微升GW4064，终浓度5-10微米（传感器饱和剂量），并等待直到传感器荧光是稳定的。 </li><li>保存实验数据导出为AVI文件。 </li><li>在打开ImageJ的两个AVI文件（通道00，​​蔚蓝，通道01，黄水晶）通过选择插件&gt;书库&gt;堆栈交织。另外，进口焦文件 （例如，.lsm或.lif文件）直接的Image J采用适当的插件（可在http://www.openmicroscopy.org），并转到步骤4.1.14。 <ol><li>对于堆栈1:使用信道01（茶晶）。 </li><li>对于栈2:使用信道00（天蓝色）。 </li></ol></li><li>点击编辑&gt;选择&gt;添加到经理，打开ROI管理器窗口。选中复选框"显示所有"。 </li><li>绘制感兴趣的少数地区（投资回报）​​，包括特定的细胞，椭圆选择工具。还绘制在一个区域中的一个圆周边的细胞或不表达的传感器，以确定背景信号的小区内。最好是绘制的ROI不非常接近在实验的细胞周长时由于焦距漂移（细胞移动在z方向上），或细胞迁移荧光强度的变化是显而易见的。 </li><li>选择一个单元格。点击插件&gt;比探查。这将导致在屏幕3:RAW，比率和Ratio_Profile。将RAW窗口显示增加黄晶（蓝线）的强度和在天蓝色（红线）的降低，如果有的FRET。的比率窗口给出了关于比茶晶/天蓝色，这将与增加的FRET增加信息。所述Ratio_Profile窗口提供在两个通道中测得的荧光强度的实际数字。如果MICROS应对安装特定的文件（ 例如，.lsm或.lif代替.AVI）文件使用的通道顺序可能发生逆转。 </li><li>从Ratio_Profile窗口附加为补充数据电子表格复制数据。做相同的所有其他细胞（和背景的投资回报率）。 注意:在在线电子表格中，所有数据被归一化到在该最大的BAS活化预期的条件。鉴于GW4064是FXR的最有力的活化剂，具有GW4064过剩温育后的荧光比设定为1。它与另外GW4064的结束所有的实验是非常重要的。这种方法的优点是，该数据是不再依赖于在不同的日子的激光强度或检测器的增益和实验，可以更容易地进行比较。此外，在表格底部的曲线图中，移动平均值可以用来平滑曲线实验噪音。然而，分析动力学数据时，由于运行AVERA不要使用此图GE还将流畅的快动力学响应。 </li></ol></li><li>使用荧光激活细胞分选的FRET测量（FACS） <ol><li>稀的所有化合物的FACS实验中无菌的FACS摄取缓冲液（0.3毫摩尔EDTA，0.5％BSA，0.01％NaN 3的和10mM D-葡萄糖）。 </li><li>收获细胞用5mM EDTA的PBS中的80％汇合的T-160个厘米2细胞培养烧瓶中。离心细胞，在250×g离心5分钟。洗涤细胞沉淀2×5毫升的FACS在RT摄取缓冲液。 </li><li>计数使用库尔特计数器或计数室细胞。 </li><li>稀粒料在FACS摄取缓冲液为1×10 6个细胞/ ml的浓度。吸液管上下以创建单个细胞的均质悬浮液。如果细胞是难以分解，把样品通过一个细胞过滤排序，以最大限度地减少喷嘴堵塞了。 </li><li>每FACS管吸取200微升细胞，并保护他们免受光。 </li><li>添加的化合物的所需的浓度（如，胆汁酸，合成FXR配体，转运蛋白抑制剂）。涡流。孵育20-30分钟在RT下振荡（在黑暗中）。 </li><li>同时，启动流式细胞仪（激光器需要时间来热身）。 </li><li>设置流式细胞门控参数实验（ 参见图3）: <ol><li>负载围绕100,000-200,000 NucleoBAS或CytoBAS转染的细胞，以确定门。 </li><li>首先调整FSC和SSC电压绘制细胞情节的中心。 </li><li>使用紫激光，调整蔚蓝（四十零分之四百五纳米）的电压值和黄水晶（二十〇分之五百二十五纳米）的电压，并确保所有NucleoBAS或CytoBAS阳性细胞散点图中绘制。 </li><li>设置正确的门（门P1达门P4）， 见图 4。 <ol><li>使用门P1通过的SSC-A / FSC-A地块中间选择的主要人群，以排除死细胞，通常显示在左下角。 </li><li>使用门P2在在FSC-H / FSC-一个窗口，从分析中除去小芯片。单细胞呈现在一个更对角线大于双峰。 </li><li>门P3的黄水晶（二十分之五百二十五纳米）/蔚蓝（四十零分之四百五NM）窗口可以用来选择NucleoBAS / CytoBAS阳性细胞，通过门控黄水晶和蔚蓝的高细胞，一个人口的右上角显示对角线剧情。 </li><li>绘制栅极P4在种群的左上角，尽可能靠近，并确保不超过5％的细胞落入本门控内。 </li></ol></li><li>加载一些未转染细胞（CytoBAS或NucleoBAS不表达），确定自体荧光。未转染的群体可以不设在栅极P3。调节栅极P3必要时要排除自体荧光的细胞。 </li></ol></li><li>将管在FACS前VORTEX样品。对于每个样品，测量至少10,000个细胞在栅极P3。 注意:人口层次窗口给出事件的数目在屏幕上显示的父栅极的每一个栅极​​和％。在门4，父的状态％细胞具有增加的黄水晶/天蓝色比率，因为所有的Nu​​cleoBAS阳性细胞的百分比。 </li></ol></li></ol>

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The authors have nothing to disclose.

这里，我们提出了详细的方案的使用一种新的遗传编码的胆汁酸传感器能够监测胆汁酸转运的时空动力学在活细胞的。该生物传感器包括稠合到FXR-LBD，从而形成基于FRET的胆汁酸传感器（BAS）和天蓝色茶晶荧光蛋白。 具有与细胞培养和FACS或（共焦）显微基本经验当胆汁酸传感器是相对简单和使用方便。然而，某些方面可能需要一些故障排除。当使用与胆汁酸转运组合胆汁酸传感器，它是用活性炭过滤的血清建议来培养细胞在培养基。木炭通过吸附从血清进一步降低胆汁酸的水平。特别是在进口如NTCP的存在，这是至关重要的，以防止在培养期间的传感器的饱和度，因为这是一个不敏感传感器的最常见的原因在实验过程中。因此，另一传感器被创建（NucleoBAS N354K / I372V）含有突变改变为胆汁酸的敏感性，创造更大的动态范围 8。定义传感器是否已经饱和，则黄水晶/蔚蓝比可以比在对照细胞不表达任何胆汁酸转运的比例，因为这些细胞被假定为具有低细胞内胆汁酸水平。另外，荧光寿命成像显微镜（FLIM）的测量，可以在执行强度无关的方式12，调查FRET。这种技术是超出了本文的范围，需要更专业的设备。其他原因的不敏感的传感器，包括使用的细胞，自体荧光和/或失去NucleoBAS表达。值得注意的是，在实验过程中的波动的天蓝色和黄晶（例如由于焦点偏移）强度可以掩盖的成像期间的FRET的变化直接可视化，而传感器的比例性质仍然允许监视胆汁酸动力学。常绝对强度的变化是温和的，用于在另外的FXR配体的个别荧光团，而增加的比率是显而易见的。 用于转染的细胞与BAS传感器FRET分析，适当的激光和过滤器应选择。茶晶强度FRET期间的增加必须与紫色二极管（405纳米）激光发射监测超过450-520（天蓝色）和520-580（黄水晶）进行测定。考虑的一个重要方面是在传感器到光漂白的敏感性。当一个或两个荧光团的强度慢慢下降时刻不添加配体，它通常表示光漂白。这种现象主要发生在激光功率太高。为了避免这种不希望的效应，激光器功率不应超过满功率的5％，并且细胞必须保持在黑暗尽可能。限制时间来寻找正确的细胞。荧光强度的波动，也可以通过在z轴焦距漂移引起的。为了对付这种情况，针孔的大小可以增加，最好是绘制的ROI不非常接近的细胞周长。 FRET的传感器，用于胆汁酸已经在多种细胞类型（U2OS，的Huh7，HepG2细胞，H69，MDCK和HEK293T细胞），其可以被转染并有稳定的表型进行了测试。然而，初级和分化的细胞，例如肝细胞难以转染，并维持稳定的特性而言。病毒转导可以帮助难以转染的细胞（构建应要求提供）。目前，我们正在产生一个鼠标线，以允许使用该传感器在新鲜分离的原代细胞分离时，可以快速去分化的。 以执行所描述的共焦实验，当务之急是所选择的细胞系是附着于培养皿和生长在一个SIngle细胞层。但是，细胞生长在悬浮液中，或贴壁细胞能够相当容易地用胰蛋白酶处理，也可以通过使用流式细胞仪进行测量。最后，建议当分析特定的传输通路，选择无内源性胆汁酸运输或合成的细胞系。 即 ，测定一定转染（突变体）转运蛋白的活性时。 所呈现的遗传编码的荧光生物传感器的BAS是用于监测单个活细胞胆汁酸转运的宝贵工具。该BAS包含FXR-LBD这是由大量的各种胆汁酸激活，允许的胆汁酸8亚细胞动力学成像。这给出了一个很大的优点相对于使用的其他技术。例如，在启动子驱动的荧光素酶报告测定中，荧光素酶信号是依赖于细胞内的记者的稳定性，不支持亚细胞的信息，并且需要的样品的破坏<s达&gt; 13。测量胆汁酸转运的另一种方法是使用荧光标记的或放射性标记的配体。然而，胆酸的荧光标记可以改变胆汁酸转运的动力学和放射性标记的胆汁酸的可用性是有限的。另外，传感器与胆汁酸转运，细胞流入和具体胆汁酸流出表达结合可以得到解决。例如，增加了共轭胆汁酸细胞内水平状TCDCA提供有关运输活动的洞察力。这也使得可能使用BAS通过使用分子或化合物抑制剂的屏幕，或检查的其他配体FXR的效果以检查具有特定的途径，例如干扰后对胆汁酸转运的影响。最后，该传感器的另一种应用是与其它荧光蛋白质进行定量分析或蛋白质的定位结合它，因为质膜转运表达和FXR激活的实例的成像在相同的细胞。

荧光共振能量转移（FRET）被广泛地用于获得与高时空分辨率的1活细胞更好地了解细胞功能。在FRET，从激发供体荧光团的能量被转移到受体荧光团。 FRET效率强烈依赖于供体和受体荧光团和它们的取向之间的距离，因此，影响的两种荧光团的构象变化敏感的读出。这一现象被利用，以产生基于FRET的生物传感器为小分子成像。改变它们的浓度可以监测作为增加/减少在所述受体与供体荧光团2的发光强度的比率。例如，基于FRET的钙生物传感器允许在活细胞中3快速和稳定的检测的游离钙离子浓度。对基于FRET的生物传感器的其它优点是成像在单个活细胞，T继承人的非侵入性，他们有能力将针对不同类型的细胞和细胞室4。 细胞内胆汁酸动力学的许多方面仍然知之甚少。例如，鲜为人知的是，游离态和结合胆汁酸转运的机制基础调节。现有的技术来监测这种运输主要是利用荧光素酶为基础的记者，放射性标记的胆汁酸，或荧光胆汁酸类似物。后者需要修改的胆汁酸，可能影响其性能。荧光素酶为基础的记者有时间分辨率差。此外，这些技术导致样品损失，是不适用的成像在单个细胞。因此，这将是有益的使用方法，使转运活性的活单细胞成像使用FRET生物传感器，特别是因为它包括比例检测5,6的优点。虽然CF变种P / YFP形式最经常使用的FRET对，采用魔橙和mCherry携带自联合诱导突变已导致FRET工具箱新颖传感器，包括一个红移胆汁酸传感器 7的膨胀的新策略。 我们以前创建的基因编码FRET胆汁酸传感器（BAS），它由一个供体荧光团（天蓝色）和受体荧光（黄水晶）的融合与法尼醇X受体（FXR）配体结合结构域（FXR-LBD）和含有肽的LXXLL模8。这与FXR-LBD肽同伙在胆汁酸依赖性。一旦FXR激活，柠檬色和蔚蓝之间的距离将改变由于构象变化。在哺乳动物细胞系，FXR激活导致在茶晶/天蓝色的比例清楚地检测到增加，而纯化的传感器的工作原理在相反的方向，并导致在FXR活化降低的FRET比率。该传感器（CytoBAS）允许监测细胞内胆汁酸的动态。由羧基末端加成亚细胞靶向基序下，BAS构建体可以靶向至核（NucleoBAS）和过氧化物酶体（PeroxiBAS），允许胆汁酸的浓度在不同的细胞区室的测量。虽然增加了过氧化物酶体靶向母题不会损害其回应胆汁酸，细胞渗透性FXR配体没有引起任何FRET内过氧化物酶体8 PeroxiBAS的变化。作为该差异的性质是未知的，该协议如下聚焦在CytoBAS和NucleoBAS。 使用这种基因编码FRET传感器的最近表现在含肝胆汁酸转运体的Na + /牛磺胆酸钠共转运多肽（NTCP）和有机溶质转运α/β（OSTαβ）8个单元 。 NTCP是主要的肝胆汁酸进口商和OSTαβ是基底肠胆酸转运体可作为进口商和出口商依赖于电化学胆汁酸浓度梯度9，10个功能两者。最近的数据表明，在由NTCP和/或OSTαβ胆汁酸运输，在FRET比率作为配体-FXR-LBD相互作用的结果健壮和快速的响应可以被观察到。 在这里，我们将详细介绍协议的方法来测量FRET如共聚焦显微镜分析和荧光激活细胞分选（FACS），突出关键环节，解决潜在的问题，并讨论替代方法。使用该基因编码的FRET传感器，与FXR-LBD胆汁酸相互作用可以量化，并直接在活细胞中监测并提供了一​​种快速，简单的可视化胆汁酸运输和动态实时的方法。编码CytoBAS和NucleoBAS哺乳动物表达质粒是市售的。因此，该生物传感器可进一步向了解胆汁酸转运或激活FXR，并提供了一​​个深入了解胆汁酸生物学和信号传导的化合物。

<table><tbody><tr><td>CytoBAS&amp;nbsp;</td><td>Addgene</td><td>62860</td><td></td></tr><tr><td>NucleoBAS&amp;nbsp;</td><td>Addgene</td><td>62861</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagles media (DMEM)</td><td>Lonza</td><td>BE12-614F</td><td>High glucose without L-glutamine</td></tr><tr><td>Penicillin-Streptomycin (pen/strep)</td><td>Lonza</td><td>17-602E</td><td></td></tr><tr><td>L-glutamine (200mM)</td><td>Lonza</td><td>17-605E</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>102-70</td><td></td></tr><tr><td>Trypsin-EDTA (10x)</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>T-25 cell culture flask</td><td>VWR international</td><td>392-0253</td><td>Laminin coated</td></tr><tr><td>T-175 cell culture flask</td><td>VWR international</td><td>392-0238</td><td>Laminin coated</td></tr><tr><td>6-well plate</td><td>VWR international</td><td>734-0229</td><td>Poly-L-lysine and Laminin coated</td></tr><tr><td>10 cm dish</td><td>VWR international</td><td>392-0243</td><td>Laminin coated</td></tr><tr><td>Diethylaminoethyl (DEAE) - Dextran</td><td>Sigma-Aldrich</td><td>D9885</td><td></td></tr><tr><td>Polyethylenimine (PEI)&amp;nbsp;</td><td>Brunschwig</td><td>23966-2</td><td></td></tr><tr><td>G418 (geneticin) 50 mg/ml</td><td>Invitrogen</td><td>10131-027</td><td></td></tr><tr><td>Hygromycin B, 50 mg/ml</td><td>Invitrogen</td><td>10687-010</td><td></td></tr><tr><td>Cloning cylinder (6 x 8 mm)</td><td>Bellco</td><td>2090-00608</td><td></td></tr><tr><td>L-15 Leibovitz culture medium</td><td>Invitrogen</td><td>21083-027</td><td>No phenol red</td></tr><tr><td>Polystyrene round bottom tube (5 ml) Facs tube</td><td>Falcon BD</td><td>352008</td><td>No cap, non-sterile</td></tr><tr><td>Falcon 2,063 tubes (5 ml)</td><td>Falcon BD</td><td>352063</td><td>Snap cap, sterile</td></tr><tr><td>Nunc Lab-Tek 8 well coverglass</td><td>Thermo scientific</td><td>155409</td><td>Sterile</td></tr><tr><td>Charcoal-filtered FBS</td><td>Life technologies</td><td>12676011</td><td></td></tr><tr><td>GW4064</td><td>Sigma-Aldrich</td><td>G5172</td><td></td></tr><tr><td>TCDCA</td><td>Sigma-Aldrich</td><td>T6260</td><td></td></tr><tr><td>CDCA</td><td>Sigma-Aldrich</td><td>C9377</td><td></td></tr><tr><td>Other chemicals</td><td>Sigma-Aldrich</td><td>n.v.t.</td><td></td></tr></tbody></table>

real time monitoring of intracellular bile acid dynamics using a genetically encoded fret-based bile acid sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

该FRET-BAS传感器提出是基于连接到两个荧光黄和天蓝色）和LXXLL模配体结合FXR（LBD-FXR）的域名。这个传感器允许在活细胞中的高空间和时间分辨率（图1的 ）调查胆汁酸运输。在天蓝色和黄水晶突变施加到促进分子内复合物（ 图 1B）的形成。胆汁酸和其它FXR配体结合到FXR，从而通过构象变化改变柠檬色和蔚蓝之间的距离。在活的哺乳动物细胞，这导致增加的FRET效率（茶晶增加，天蓝色减少）（ 图 1C，1D）。在活细胞定量强度为基础的FRET实验的共聚焦显微镜，我吨观察到牛磺鹅去氧酸（TCDCA）处理（30μM）表达NucleoBAS缺乏胆汁酸转运不显示在FRET任何改变U2OS细胞，而平均茶晶/天蓝色的比例与GW4064（5微米）（图治疗后显着增加1 C）。与此相反，U2OS细胞共表达NucleoBAS，NTCP和OSTαβ确实显示在加入TCDCA的明显增加在柠檬色/天蓝色比，和加法GW4064（图1（D））后更大的增加比率。这表明TCDCA是不能穿过细胞膜并需要特定的转运进入细胞。 传感器的细胞定位是通过特异性定位信号的存在来确定。 NucleoBAS靶向细胞核的核定位信号（图&lt;强&gt; 2 B），而CytoBAS不包含任何位信号，因此保持在细胞质中（图2 的A）。生物传感器也可以与其他的荧光蛋白的显像联合，使蛋白质表达/本地化和FXR激活的测量中相同的细胞。 例如 ，图2 的C显示 U2OS细胞共转染NucleoBAS和NTCP-mKate2。另外，传感器还可以与其他传感器，用于多个参数的亚细胞成像同时结合。例如，NucleoBAS用TGR5（EST克隆IMAGE＃5221127）和cAMP的胞质传感器（T EPAC VV）11的同时，测量TGR5和FXR激活在同一细胞中表达的沿。一旦TGR5结合，cAMP水平在其通过cAMP的传感器检测出的胞质溶胶增加，而FXR激活在细胞核是可见同时ualized。 图 2D-F &#160;示出了时间序列处理后由NucleoBAS-TGR5-ŤEPAC 节细胞6共焦图像用GW4064（5微米）（图2 D）的 ，具有TCDCA（10μM）（图2 E）或与鹅脱氧胆酸（CDCA） （10μM）（图2 F），在t = 0的绿色颜色代表其中四百五十零分之五百二十五nm发射率降低的区域（表示cAMP的海拔）和粉红颜色代表的增加发射系数（FXR激活），相比比率在实验开始。 此外，流式细胞仪可用于筛选细胞上单细胞水平或对整个人口的高通量筛选的池。 U2OS细胞表达NucleoBAS是EXCIT编带紫405nm的激光器和荧光收集在四十分之四百五范围为天蓝色和二十〇分之五百二十五范围为黄晶。为了改善的基于FACS的FRET实验的效率，适当的门必须被设置（图3）。在P1门排除细胞碎片和P2门从分析中删除小芯片。接着，将分析限制为黄晶（激发488激光）和天蓝色（激发405激光）阳性细胞通过栅极3.最后，栅极P4表示细胞具有高黄水晶/天蓝色发光比率的百分比（使用的天蓝色激发与405 nm激光）。对于每个样品，至少10,000个细胞的内门3下降进行测定。 随后，基于FACS的FRET流式细胞仪被用来计算后的30分钟温育与TCDCA和GW4064增加FRET在活细胞中。表达NucleoBAS未处理的U2OS细胞表现出&lt;5％的细胞在门P4（黄水晶/ Cerulean高的细胞）。在没有任何胆汁酸转运体，类似的比例，观察30μMTCDCA处理的细胞（ 图 4 A）。与此相反，细胞共表达NucleoBAS，NTCP和OSTαβ确实表现出大约38％的黄晶/天蓝色高细胞的量增加。加入10μMGW4064后，细胞中几乎90％是黄水晶/蔚蓝高不论OSTαβ或NTCP（ 图 4 B）的表达。数据可以呈现在条形图（％细胞门4，人口4）（图4 C，D）或作为黄水晶-蔚蓝比（图4，E，F）人口直方图。 <img alt="图1" src="/files/ftp_upload/53659/53659fig1.jpg" /> F胆汁酸传感器（BAS）的 igure 1。设计 （一）遗传编码的胆汁酸传感器的作用方式的结构示意图。它由一个供体荧光团（天蓝色）和受体荧光（黄水晶）通过FXR-LBD连接和融合到FXR辅因子肽（LXXLL模）的。 （ 二）BAS示意图域架构。该Q204F / V224L荧光基因突变促进天蓝色和黄水晶的分子相互作用形成。所述NucleoBAS构建体含有一个c-末端核定位信号（NLS），因此积累在细胞核中，而CytoBAS构建体缺乏任何击靶序列，因此，示出了细胞溶质定位。 （ 三）代表共焦为基础的FRET实验BAS作为一种工具来监视结合胆汁酸转运。在黄水晶/蔚蓝比没有变化后，30μMTCDCA除了在细胞中只EXP观察ressing NucleoBAS。当5微米GW4064加入，强增加黄水晶/蔚蓝的比例进行了观察。 TCDCA的（D）导入（30μM）导致在NTCP和OSTαβ共转染的细胞相当激活传感器。随后GW4064（5μM）处理导致在黄水晶/天蓝色的比率进一步增加。结果以平均值±标准差，N = 6单个细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/53659fig1large.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/53659/53659fig2.jpg" /> 图 2。 本地化FRET-BAS构建在活细胞中。U2OS细胞转染CytoBAS（A）或 NucleoBAS（B）的共聚焦显微镜图像。 （C）的U2OS细胞共移植sfected与NucleoBAS和NTCP-mKate2。比例尺条为10微米。 （D - F）时间序列NucleoBAS-TGR5- 牛逼 EPAC VV转染的细胞。 （D）GW4064激活FXR（增加450分之525纳米比），但并不TGR5。 （E） - TCDCA激活TGR5在质膜上（降低450分之525纳米比），但不能够进入细胞激活FXR在细胞核中。 （F）CDCA激活TGR5在质膜以及FXR在细胞核（增加核四百五十零分之五百二十五纳米的比例，在细胞质中减少）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/53659fig2large.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/53659/53659fig3.jpg" /> 图 3。 流式细胞仪门parameteRS（一）在SSC-A / FSC- 窗口中的第1页门用于排除死细胞。 （二）在FSC-H / FSC-A的窗口，P2的门被吸引到消除分析双峰事件。 （C）在（二十〇分之五百二十五纳米）/蔚蓝（四十〇分之四百五十○nm）的窗口，NucleoBAS（和CytoBAS）阳性细胞被选中的黄水晶（门P3）。 （四）最后，在黄水晶（二十分之五百二十五纳米）/蔚蓝（四十分之四百五十〇纳米）窗口中，P4门包含FACS实验茶晶/蔚蓝高细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/53659fig3large.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/53659/53659fig4.jpg" /> 图 4。 代表FACS实验测量用NucleoBAS胆汁酸转运。（A </strong<html> &gt;，B）的FACS-曲线示出的量茶晶/天蓝色高活NucleoBAS表达U2OS细胞（A）和U2OS细胞共表达NucleoBAS，OSTαβ和NTCP（B）中，与对照缓冲液（左），30μM处理后TCDCA（中）或10μM的GW4064（右）。 （C，D）柠檬色/天蓝色高细胞后30分钟孵育TCDCA或GW4064在U2OS细胞表达NucleoBAS和酒吧百分数图共转染（D）或无（C）的OSTαβ和NTCP。 （E，F）柱状图显示的黄水晶/蔚蓝比U2OS细胞后30分钟孵育TCDCA（黄色）表示NucleoBAS带或不带OSTαβ和NTCP，GW4064（红色）或对照缓冲液（蓝色）的人口分布。所有条件测定三次。条形代表平均值±标准差（SD）（N = 3）。TTPS://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/53659fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 补充文件1: 模板BAS一般 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/Supplemental_File_1._template_BAS_general.xls" target="_blank">请点击此处下载该文件。</a> 补充文件2: 模板BAS蔡司徕卡导入的数据 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53659/Supplemental_File_2._template_BAS_Zeiss_Leica_imported_data.xls" target="_blank">，请点击此处下载该文件。</a>

Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

real-time-monitoring-intracellular-bile-acid-dynamics-using

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.8K 次观看.  Academic Medical Center. We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types. 

视频: Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

科研

生物工程

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

谷氨酰胺通量成像使用基因编码的传感器

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

使用基于 BRET 的方法在活细胞中进行 CRAF-14-3-3 相互作用分析

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

作者聚焦：整合基于 BRET 的检测和罕见突变分析以破译活细胞中的 RAF 激酶调控

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

生物化学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

生物学

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

医学

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

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 ...

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

摘要

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成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

FIBS功能的无创性代谢轮廓

谷氨酰胺通量成像使用基因编码的传感器

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

使用多氟化胆汁酸和

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

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

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

摘要

探索更多视频

此视频中的章节

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

FIBS功能的无创性代谢轮廓

谷氨酰胺通量成像使用基因编码的传感器

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

使用多氟化胆汁酸和

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

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

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用