这项工作是由美国国立卫生研究院授予1R21NS064412，NSF资助1052048和Jeffress纪念信托基金资助的J-908的支持。

1，样品制备注:在许多情况下灌注实验洗去细胞的显著部分，它可以成为一个令人沮丧的问题。虽然没有必要对所有的细胞系，涂层覆盖玻璃表面用聚-L-赖氨酸（添加0.01％的溶液1.0 ml/25厘米2的表面，孵育&gt; 5分钟，用细胞培养级水洗涤两次，干燥生物安全柜）提高细胞的粘附。同时，要注意使用的细胞系的生物安全水平（BSL）的，并按照批准地方环境健康和安全办公室的标准作业程序。在该实验中，转染的COS7细胞用于由于低内源性谷氨酰胺转运活性（参见图4）。 <ol><li>种子COS7细胞在〜在Dulbecco改良的Eagle培养基（DMEM）70％-80％汇合度+10％宇宙的小牛血清和100 U / ml青霉素/链霉素，无论是在无菌25毫米盖玻片放置在6孔培养菜或8孔玻璃底的幻灯片。在37℃，5％CO 2，100％湿度下24小时。 </li><li>更换生长培养基，以DMEM +10％宇宙的小牛血清（不含抗生素），根据制造商的方案。成长O / N在37℃，5％CO2，100％湿度。 </li><li>将每个质粒DNA的0.4毫克编码谷氨酰胺传感器（染pcDNA3.1，携带FLIPQTV3.0传感器在CMV启动子10）和的mCherry标记ASCT2 10至50ml无血清培养基（OPTI-MEM），轻轻混匀。 </li><li>加入1 ml转染试剂至50ml无血清培养基。在室温下孵育5分钟。 </li><li>混合含有DNA（步骤1.3）和转染试剂（步骤1.4）的两种溶液并孵育20分钟。 </li><li>轻轻地加在细胞上的顶端的转染试剂和由摆动腔轻轻混匀。孵育24小时后，在37℃，5％CO 2，100％湿度。 </li><li> 24小时后，更换培养基，以DMEM +10％宇宙小牛血清（CCS）+ 100单位青霉素/链霉素。 </li><li>细胞成像48-72小时后转染。 </li></ol> 2，灌注实验注意:对于在该实验中使用COS7细胞，灌注介质和腔室保持在室温和环境CO 2浓度。然而，如果正在使用的电池需要较高的温度和CO 2浓度控制为生存，加热的显微镜载物台和/或环境室应该被使用。 <ol><li>准备灌注缓冲自动对焦（见材料清单）。附加旋塞至50ml注射器，关闭旋塞然后填写与灌注液。打开水龙头一个短暂的时期，以填补与缓冲活塞的头部空间。 </li><li>切换的8通道，重力进料灌注系统与灌注铅笔，然后选择在选择功能选项"手动模式"。放置一个废物容器下的灌注铅笔。 </li><li>手动通过按下相应的数字激活的端口，用10 ml注射器含有水补油管，然后关闭该端口。一旦端口被装满了水，设置包含缓冲区自动对焦灌注系统的端口1-6的注射器，然后打开旋塞。再次打开的端口，以取代水与缓冲管。流速应为约0.8毫升/分钟。 </li><li>按取消一次灌注控制器返回到"选择功能"菜单。切换到"编辑程序"选项，然后在灌注协议表1所示。保存的灌注程序。 </li><li>取出来的细胞培养箱中。与灌注缓冲液洗两次，然后将细胞在舞台上。灌注系统连接到室内。如果一个开放室使用，另设泵，去除室内的灌注液。 </li><li>打开Slidebook软件。开始一个新的幻灯片。 注意:下面的过程是特定于Slidebook软件，但其他软件如MetaFluor和萨 - 要素也可用于此处所述的成像的类型。理论上实验可以使用允许时间 - 过程成像任何软件来执行，并且图像序列可以被分析后的实验使用软件如ImageJ的（http://rsbweb.nih.gov/ij/）或斐济（呻/ fiji.sc /斐济）。然而，软件，允许的受体/供体比实时监测是非常有用的。 </li><li>安装滑道到荧光显微镜阶段。发现细胞共表达的传感器和ASCT-mCherry的使用适当的过滤器集（请参阅材料清单）下的"聚焦"功能。通过点击"相机"选项卡和"收益"项下滑动滑动条调节增益。此外，从调节中性密度滤镜下拉菜单中的中性密度滤镜设置。注意:如果过滤器多维数据集不包括眼滤镜，不要用眼睛片，因为肖尔的T波长的激发光对眼睛有害。使用电脑屏幕找到细胞来代替。 </li><li>获得的细胞图像with donor ( mTFP1) ex donor(mTFP1) em , donor ( mTFP1) ex acceptor(venus) em and acceptor ( mTFP1) ex acceptor(venus) em m channels （过滤器在材料列表中指定）。单击"图像捕捉"，然后在采集窗口，指定将要使用的过滤器。点击"测试"按钮，通过在框中键入所需的曝光时间旁边的过滤器设置来调整曝光时间。注:所有三个通道需要被暴露于相同的长度。当调节摄像参数，取预期的FRET效率的变化和折叠增加考虑:例如，如果预期的FRET效率在实验过程中向上去2倍，摄像参数应调整从而使供体的前接受器烯在静息状态下的强度应&lt;可被仪器记录在最大值的50％，从而使信道不会成为在实验过程中达到饱和。从标记细胞信号应至少比背景区域高3倍。 </li><li>使用该软件区域工具绘制一个地区的利益。可替换地，软件功能来选择上述一定强度的像素都可以使用，然后转换成的区域。要执行此操作，请单击面膜 - 分部，然后移动滑动条来突出显示所需的区域。 </li><li>选择在拍摄类型框中的"时间流逝"，然后指定时间间隔（10秒在这个实验）和实验时间点。 </li><li>在一个区域画出一个区域，没有任何荧光细胞。这个区域是用于背景减法。用鼠标右键单击绘制的区域，然后将其设置为背景。注意:在理想情况下，细胞是不吨表达该传感器应作为背景区域占两者的自体荧光和照相机所检测到的背景荧光。如果没有这样的细胞可在视场中，至少在没有细胞的区域应该被用来解释背景荧光。 </li><li>选项​​ - 在"运行程序选择功能"选择所需的程序。切换到保存的程序（见步骤2.3），然后打的灌注控制器上的ENTER键。现在，这个程序被加载，并且击中任何键将开始灌注。 </li><li>确保灌注和成像实验开始的同时按一下在同一时间按下灌注控制器上的按键，在拍摄窗口中的"开始"按钮。 注:建议记录那里的解决方案进行了交流（这可以通过使用"票据"功能，采集选项卡中找到来完成）的时间点。 </li><li>执行相同的灌注协议USI纳克细胞表达，预计将是饱和的或者未结合的实验条件下的传感器。 注意:在这里，FLIPQTV3.0_1.5μ，这是在实验条件下饱和的，被用作对照， 图6这样的对照实验是必要的，以确认该FRET效率的变化是由于在基板上的实际变化，而 ​​不是。由于在其它参数，如细胞内pH的变化。 </li></ol> 3，后期实验分析<ol><li>检查如果在实验过程中发生样品漂移。绘制感兴趣区域（ROI）区域S在0时间点拍摄的图像上，然后移动在成像窗口顶部的滑动杆，并检查电池掉出的ROI在后来的实验中拍摄的图像。 </li><li>如果是的话，那么使用跟踪功能来校正漂移，首先，在细胞周围建立口罩通过选择面膜 - 分类，然后将它指定截止到HIG行hlight区域含有细胞。追踪区域单击面膜 - 粒子跟踪 - 基本粒子跟踪。 </li><li>必要时重新绘制的ROI和背景区域。 </li><li>导出的感兴趣区的平均强度比实验的过程。点击统计 - 出口 - 比/慢速拍摄数据。 </li><li>当底物浓度的FRET效率和量化是没有必要的，画出作为基材的动态代理的强度比（ 供体前接受器烯 ，/供体的前施主烯 ）的时间过程。以确定细胞内浓度，计算出的最大和最小的比率变化（ 即，在所述基板的饱和和不存在，分别），ΔR 最大 - ΔR 分钟 ，由Δratio（ΔR）与下面的等式的非线性回归: ΔR= [S] /（K D + [S]）×（ΔR <suB&gt;最大值- ΔR 分钟 ）+△ 分钟 其中[S]是底物浓度在胞质溶胶中。使用ΔR 最大 -传感器得到ΔR 最小值 ，饱和度（S）在给定的ΔR被计算为 S =（ΔR - ΔR 分钟 ）/（ΔR 最大 - ΔR 分钟 ） S可进一步被转化成使用以下等式的底物浓度[S]: S = [S] /（K D + [S]） 注: 接受器前受体EM信道的强度应同时绘制研究实验条件是否影响受体的直接荧光。 </li><li>当基线漂移由于光漂白观察，进行基线校正。要做到这一点，画出供体和受体随着时间的强度（ 图5A）。然后确定用于日的时间点ë基线，根据在灌注系统中的延迟和特定的细胞（ 图5B）的转运活性。 </li><li>计算多项式拟合最能说明基线。然后，归一化的原始强度从每个时间点与基线（ 图5C）。从标准化的供体和受体的强度计算强度比（ 捐赠前受体他们，/捐赠者捐赠者除外EM）。 </li></ol>

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没有什么可以透露。

成像实验的成功取决于几个关键因素。其中一个因素是传感器的使用，如上面所讨论的亲和力。在感兴趣的亚细胞区室中衬底的绝对浓度，但是，常常是未知的。因此，我们建议您尝试多种传感器与交错的亲和力，以找到一个所需的实验条件下效果最好。例如，在我们的情况下，我们转染的COS7细胞中谷氨酰胺传感器，1.5μ，100μ，2分，和8米（ 图3和数据未显示）。 另一个重要因素是传感器的蛋白质的表达水平。需要用于检测表达水平之间，实验而变化，例如，当一个短暂的事件（ 例如，单个动作电位）需要被观察到的更高的表达水平可能成为必要19。因此，在大多数情况下，所要求的电平必须是empiricallŸ确定。如果目标存在于非常低的浓度在细胞中，由于目标耗竭内源性处理的扰动可以成为一个问题20。一个独立的检测，以评估这种可能性，如果可用，因此需要。在这篇文章中所示的实验中，由CMV启动子驱动的表达被认为是足够的。在情况下的启动子具有弱得多的活动需要使用时，调整以增加信号强度可能变得必要。例如，在曝光期间的信号强度和光致漂白之间折衷需要达到。除了较长的曝光时间，可以改变因素，如荧光团的亮度，传感器，分块的最大强度的变化，和CCD摄像机的灵敏度，以提高信号的强度。 当没有比变化在实验条件下观察到即使上述两个条件都可能得到满足，这是可能的，对于给定的代谢物或离子的细胞体内平衡是足够强的以掩盖转运活性（参见结果部分）。在这种情况下，细胞系具有不同的生化活性，可能需要作为背景来检测转运活动10，21。 虽然这些传感器允许一个监控底物浓度动力​​学在更高的时空分辨率比提取为基础的方法，测定绝对浓度需要进一步的实验步骤和注意事项。通常，底物浓度，计算时假设传感器的亲和力，通常是通过滴定纯化的蛋白质传感器计算出的，当在细胞中表达是不变的。浓度是使用以下单一结合等温线计算的， [S] = K 深x（R - R 分钟 ）/（R &lt;/ em&gt;的最大值 - R） [S]是底物浓度，K d是解离常数在体外测定中，r是在给定时间点所观察到的受体/供体比，R最小值是受主/施主时比传感器是在载脂蛋白形式，且r 最大是受主/施主时，比所有传感器被绑定到衬底上。R 最小 和 r 最大值由参数如传感器的浓度，所使用的成像设置，蜂窝milleu的组合物，并且因此需要在原位被测定的影响要确定R 最小 和 r 最大值 ，实验条件，使细胞内底物浓度的修改成为必要。例如，有选择性的离子载体（ 例如，离子霉素的Ca 2 +），22或穿孔试剂如二gitonin 23可用于平衡细胞内底物浓度与外部浓度。 其中一个在实验中该协议体现了类型的限制是低吞吐量;该分析是在细胞中的相对小的（&lt;10）的数量分析一种代谢物的限制。技术的进步正在作出，但是，为了克服这种局限性。例如，用在自动化，高含量筛选的进步，现在可以在组合与小分子或RNAi文库用遗传编码的传感器;细胞中表达的生物传感器可以在高通量的格式（ 例如，96 -或384 -孔）中生长，然后单独的孔可以与任何的siRNA或化学品处理和成像。这样的实验允许识别，破坏，可以由生物传感器24 25观察到的生物学过程的siRNA构建体或化学品。另一个令人兴奋的最新进展包裹体DES时间分辨微流体流式细胞仪，它允许高通量检测的荧光共振能量转移效率的变化在细胞水平上26的发展。这样的技术进步将有助于新的候选药物和生物过程的新组件的发现。

由于技术的显着进步，使考试的转录组和蛋白质组在细胞水平上的，它现在已经很清楚，生物化学和由此产生的通量代谢物和离子是高度细胞类型特异性。例如，在哺乳动物的肝脏，顺序谷氨酰胺降解和合成是同时进行由门静脉周围的细胞和静脉周围的细胞分别，同时消耗过量的氨在后者的1-3喂养铵到尿素循环在前者的细胞类型。在某些情况下，显著生化异质性被检测，即使在一个单一的"小区类型"4，5。除了 ​​这样的空间特异性，代谢物和离子的细胞水平是高度动态的（ 例如，信号分子，如Ca 2 +，环 ​​核苷酸）。代谢物和离子的时空模式往往能起到关键作用的信号转导。 Monitoring代谢物和离子的细胞动力学，但是，提出了独特的挑战。在许多情况下，其浓度的变化是快速和短暂的，通过信号转导分子如Ca 2 +，内〜20毫秒的树突棘6，该衰变的情况举例说明。此外，内部和之间的细胞的生化途径的分隔使得难以用萃取和柱色谱/质谱技术定量代谢物和离子的动力学。 遗传编码的传感器，用于生物分子现在广泛由于高时空分辨率，允许实验者研究短命和/或隔离的分子动力学使用（7评论，8）。这些基因编码的传感器大致可以分为两大类;基于强度的传感器和ratiometic传感器。强度为基础的传感器通常由一个结合结构域和一个感orescent蛋白（FPS），以及溶质结合的结合结构域改变的荧光强度。 Ratiometic传感器，在另一方面，往往采取的促进共振能量转移（FRET）2的FP充当FRET对的优势。这些传感器包括一个结合结构域和两个FP的，与溶质结合诱导在2的FP之间的FRET效率的变化。大量的传感器，用于生物学上重要的代谢物和离子已被开发，在过去十年中8,9。 其中由该基因编码的传感器所提供的令人兴奋的可能性是他们在膜转运过程的高分辨率分析，这在以前是不容易察觉在细胞水平上的使用。遗传编码的传感器便利的运输机制，如底物特异性和pH依赖性10，11中的分析。此外，在同遗传水库组合OURCES如RNA干扰的库构建的模式生物，它现在可以进行全基​​因组搜索使用基因编码的传感器新颖的运输过程。事实上，利用基因编码的传感器导致的以前未知转运多例12，13的发现。 最近，我们实验室已经开发出一系列基于FRET的传感器谷氨酰胺。我们已经证明，细胞谷氨酰胺水平可以使用这样的FRET谷氨酰胺传感器10被可视化。这些传感器由在C-末端glnH的（ 图1）一个FRET供体（mTFP1）插入到细菌谷氨酰胺结合蛋白glnH和FRET受体（金星）的。这些传感器中的FRET效率下降时的谷氨酰胺结合，导致受体/供体的强度比的降低。谷氨酰胺转运过程的精细调节是重要的生物过程，如neurotransmission 14,15和尿素循环的维持在肝脏1，16，17。 在这里，我们将展示分析运输活动与FRET传感器谷氨酰胺，使用宽场荧光显微镜下建立的方法。这里显示的实验的目的是检测在单个细胞中转运活动，并检查一个瞬时表达转运蛋白的底物特异性。

<table><tbody><tr><td>Inverted fluorescent microscope</td><td>Olympus</td><td>IX81F-3-5</td><td>An equivalent inverted fluorescent microscope from other suppliers would also be appropriate.</td></tr><tr><td>Excitation filter (mTFP1)</td><td>Omega Optical</td><td>3RD450-460</td><td>Band width 450-460 nm</td></tr><tr><td>Excitation filter (Venus)</td><td>Chroma</td><td>ET500/20</td><td>Band width 490-510 nm</td></tr><tr><td>Emission filter (mTFP1)</td><td>Chroma</td><td>HQ495/30m</td><td>Band width 475-505 nm</td></tr><tr><td>Emission filter (Venus)</td><td>Chroma</td><td>ET535/30m</td><td>Band width 520-550 nm</td></tr><tr><td>Dichroic mirror (FRET channels)</td><td>Chroma</td><td>470dcxr</td><td>Long pass, 470 nm cut off</td></tr><tr><td>Dichroic mirror (YFP channel)</td><td>Chroma</td><td>89002bs</td><td>Passes 445-490 nm, 510-560 nm, 590-680 nm</td></tr><tr><td>mCherry filter set</td><td>Chroma</td><td>49008</td><td>Excitation 540-580 nm, long pass dichroic with 585 nm cut off, emission filter 595-695 nm</td></tr><tr><td>Light source</td><td>Olympus</td><td>U-LH100L-3-5</td><td>LED- or halogen light source that produces stable light intensity, mercury lamps are not recommended</td></tr><tr><td>CCD camera</td><td>Qimaging</td><td>Rolera-MGi EMCCD</td><td></td></tr><tr><td>Apochromatic fluorescence objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Perfusion system, ValveBank II</td><td>AutoMate Scientific</td><td>[01-08]</td><td>Other perfusion systems that allow fast solution exchange would also work</td></tr><tr><td>Laminar-flow chambers</td><td>C&amp;amp;L Instruments</td><td>VC-MPC-TW</td><td>Other larminar-flow chambers would also work.</td></tr><tr><td>Chambered slide</td><td>Lab-Tek</td><td>154534</td><td>For open-chamber experiments</td></tr><tr><td>Perfusion pump</td><td>Thermo Scientific</td><td>74-046-12131</td><td>For open-chamber experiments</td></tr><tr><td>Software supporting ratiometric measurements</td><td>Intellegenent Imaging Innovation</td><td>Slidebook 5.5</td><td></td></tr><tr><td>Laminar flow biosafety cabinet</td><td>ESCO</td><td>LA-3A2</td><td></td></tr><tr><td>Isotemp CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td>Thermo Scientific</td><td>13-255-25</td><td></td></tr><tr><td>Dulbecco&#039;s MEM (DMEM)</td><td>Hyclone</td><td>SH30243.01</td><td></td></tr><tr><td>Cosmic calf serum</td><td>Hyclone</td><td>SH3008703</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Serum-free medium for transfection (OPTI-MEM I)</td><td>Invitrogen</td><td>31985</td><td>Used with Lipofectamine 2000</td></tr><tr><td>Poly-L-Lysine solution</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>25 mm circular glass cover slips</td><td>Thermo Scientific</td><td>12-545-102</td><td>In case VC-MPC-TW is used</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668027</td><td>Other transfection reagents can also be used.</td></tr><tr><td>Solution A (Hank&#039;s buffer)</td><td></td><td></td><td>9.7 g HANK salt (Sigma H1387), 0.35 g NaHCO&lt;sub&gt;3&lt;/sub&gt;, 5.96 g HEPES to 1 L, pH adjusted to 7.35 with NaOH</td></tr><tr><td>Solution B</td><td></td><td></td><td>Solution A + 0.04 mM Gln</td></tr><tr><td>Solution C</td><td></td><td></td><td>Solution A + 0.2 mM Gln</td></tr><tr><td>Solution D</td><td></td><td></td><td>Solution A + 1 mM Gln</td></tr><tr><td>Solution E</td><td></td><td></td><td>Solution A + 5 mM Gln</td></tr><tr><td>Solution F</td><td></td><td></td><td>Solution A + 5 mM Ala</td></tr></tbody></table>

glutamine flux imaging using genetically encoded sensors

遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。一个巨大的品种，例如传感器，生物分子在过去15年中面世，其中一些成为了经常被用在许多实验室不可缺少的工具。 其中一个基因编码传感器的令人兴奋的应用之一是在调查细胞转运过程中使用这些传感器。转运如动力学和底物特异性的属性可以在细胞水平进行调查，为运输活动的细胞类型特异性分析的可能性。在这篇文章中，我们将演示如何转运动力学可以用基因编码的谷氨酰胺传感器为例进行观察。实验设计，实验设置的技术细节，和考虑后的实验分析将进行讨论。

典型时程实验示于图2，在这些实验中，FRET谷氨酰胺传感器为8毫米（FLIPQTV3.0_8m， 图2A和2B）和100μM（FlipQTV3.0_100μC和D）的亲和性被共表达以强制性氨基酸器ASCT2 18 COS7细胞10。谷氨酰胺流入被检测作为给体（mTFP1）和受体（金星）（ 图2A和2C）之间的荧光强度比的变化。谷氨酰胺在另一衬底（丙氨酸）的存在下的流出也清楚地表明在这些实验中。用两个传感器，归一化的荧光强度变化可往复运动（ 即，供体强度上升作为受体强度下降， 如图2B和2D）在底物的存在，这表明所观察到的比率变化确实是由于吨o在荧光共振能量转移效率的变化。这些实验表明，谷氨酰胺的浓度在这些细胞中的波动非常动态的实验条件下;从100μM的传感器的检测范围内的近饱和浓度为8毫米的传感器。 转运蛋白的底物特异性，也可以使用传感器，在图3中展示了检验。在这些实验中的细胞预装载有谷氨酰胺，然后各种氨基酸加入到细胞外灌注液，以检查这些氨基酸是否能诱导谷氨酰胺流出。正如预期的那样，ASCT2基板（丙氨酸，丝氨酸，半胱氨酸，苏氨酸，D-丝氨酸）诱导谷氨酰胺流出（ 图3A），而非基底氨基酸（Pro中，组氨酸，赖氨酸）没有（ 图3B），证实与以往的研究18。通常，底物特异性是由竞争测定法使用具有竞争性底物混合，放射性同位素标记的底物测得，这是相当费力的，需要的细胞群被均匀地表达转运进行研究。光学成像在这里体现提供了一种替代方法。 当没有FRET效率的变化都在加入底物观察到的，有几个原因可以考虑。一个可能的原因是低吸收能力被测试的底物和/或保持在细胞内的浓度酶的高活性。例如，谷氨酰胺浓度的变化是最小的在COS7细胞中不进行测试的条件下表达ASCT2转运体，即使该细胞系可以清楚地生长在媒体，其中谷氨酰胺在主要氮源（ 图4）。此外，如果传感器的亲和力过高或过低相比，细胞内的浓度，大多数传感器蛋白质仍将组成性结合或未结合分别;因此没有FRET效率的变化将是detected。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig1highres.jpg" width="500" /> 图1:配置一个FRET谷氨酰胺传感器（A）开（青色）和关闭glnH（黄色）构象，谷氨酰胺大肠杆菌结合蛋白。其中mTFP1插入的位置被标记为洋红色。FLIPQTV_3.0传感器（二）示意图。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig2highres.jpg" width="500" /> 图2:使用FLIPQ-TV3.0_8m和100μ传感器在体内谷氨酰胺测量。 （一）COS7细胞的venus/mTFP1比例共表达FLIPQ-TV3.0_8m传感器和ASCT2-mCherry的。的mCherry标记用于鉴定表达所述传输的细胞呃不干扰的mTFP1或金星发射通道。将细胞灌注HEPES缓冲的Hank氏缓冲液（pH 7.35）。当细胞外谷氨酰胺（红色）和丙氨酸（蓝色）加入到灌注介质的时间点被表示为方块图所示。实线和虚线代表在同一实验中测得两个单独的电池（B）供体（mTFP1）和受体（金星）通道中（A）所示的实验的强度。的值进行校正为光漂白和归一化到基线（C）和（D）一个类似的实验中（A）和（B）中，用表达FLIPQ-TV3.0_100μ传感器COS7细胞中进行。 （图10修改）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig3highres.jpg" width="500" /> 图3。消除细胞谷氨酰胺吨hrough中的外部氨基酸存在下的ASCT2转运体，可视化使用FLIPQ-TV3.0_8m传感器。 （A）的胞质谷氨酰胺通过加入外丙氨酸，丝氨酸，半胱氨酸，苏氨酸和D-丝氨酸的出口。当细胞外谷氨酸（红色框）或其它氨基酸（蓝色框）被添加到媒体灌注时间点都显示为方块图形上方。（b）增加临，赖氨酸，他（黑盒）不会改变细胞内谷氨酰胺浓度，而另外丙氨酸（蓝色框）的促进谷氨酰胺的出口。实线和虚线代表在同一​​实验中测得两个单独的电池。所有氨基酸均在5 mm外部浓度添加。 （图最初发表于10）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig4highres.jpg" width="500" /> 图4。COS7细胞venus/mTFP1比expressing FLIPQ-TV3.0_8m传感器（A）和100μ传感器（B）将该细胞灌流HEPES缓冲的Hank氏缓冲液中。当细胞外谷氨酰胺（5毫摩尔）加入到灌注介质的时间点被表示为红色方块图所示。实线和虚线代表在同一​​实验中测得两个单独的电池。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig5highres.jpg" width="500" /> 图5。校正为光漂白（A）从图2A和图2C所示的两个单元上游强度（B）被选择为计算基准那个数据点。多项式拟合曲线如图所示。（C）强度在A中所示的渠道，规范化的反对B.计算基准本图中所使用的数据集是IDEntical到图2A和图2C所示。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51657/51657fig6highres.jpg" width="500" /> 图6。使用FLIPQ-TV3.0_1.5m传感器在体内谷氨酰胺的测量（A）转染的COS7细胞的venus/mTFP1比共表达FLIPQ-TV3.0_1.5m传感器和ASCT2-mCherry的。的mCherry标记用于鉴定表达本转运无干扰的mTFP1或金星发射通道的细胞。将细胞灌注HEPES缓冲的Hank氏缓冲液（pH 7.35）。当细胞外谷氨酰胺（红色）和丙氨酸（蓝色）加入到灌注介质的时间点被表示为方块图所示。实线和虚线代表在同一实验中测得两个单独的电池（B）供体的强度（mTFP1）和受体（金星）频道在（A）所示的实验。的值进行校正为光漂白和归一化到基线。 <table border="0" cellpadding="0" cellspacing="0" width="474"><tbody><tr><td>时间（min） </td><td>溶液A </td><td>溶液B </td><td>溶液C </td><td>溶液D </td><td>溶液E </td><td>溶液F </td></tr><tr><td> 0 </td><td>阀门上</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td>阀门关闭</td><td>阀门上</td><td></td><td></td><td></td><td></td></tr><tr><td> 4 </td><td>阀门上</td><td>阀门关闭</td><td></td><td></td><td></td><td></td></tr><tr><td> 6 </td><td>阀门关闭</td><td></td><td></td><td></td><td></td><td>阀门上</td></tr><tr><td> 8 </td><td>阀门上</td><td></td><td></td><td></td><td></td><td>阀门关闭</td></tr&gt; <tr><td> 10 </td><td>阀门关闭</td><td></td><td>阀门上</td><td></td><td></td><td></td></tr><tr><td> 12 </td><td>阀门上</td><td></td><td>阀门关闭</td><td></td><td></td><td></td></tr><tr><td> 14 </td><td>阀门关闭</td><td></td><td></td><td></td><td></td><td>阀门上</td></tr><tr><td> 16 </td><td>阀门上</td><td></td><td></td><td></td><td></td><td>阀门关闭</td></tr><tr><td> 18 </td><td>阀门关闭</td><td></td><td></td><td>阀门上</td><td></td><td></td></tr><tr><td> 20 </td><td>阀门上</td><td></td><td></td><td>阀门关闭</td><td></td><td></td></tr><tr><td> 22 </td><td>阀门关闭</td><td></td><td></td><td></td><td></td><td>阀门上</td></tr><tr><td> 24 </td><td>阀门上</td><td></td><td></td><td></td><td></td><td>阀门关闭</td></tr&gt; <tr><td> 26 </td><td>阀门关闭</td><td></td><td></td><td></td><td>阀门上</td><td></td></tr><tr><td> 28 </td><td>阀门上</td><td></td><td></td><td></td><td>阀门关闭</td><td></td></tr><tr><td> 30 </td><td>阀门关闭</td><td></td><td></td><td></td><td></td><td>阀门上</td></tr><tr><td> 32 </td><td>阀门上</td><td></td><td></td><td></td><td></td><td>阀门关闭</td></tr><tr><td> 34 </td><td>阀门关闭</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1实施例在本实验中所用溶液A的灌注协议:9.7克HANK盐（H1387），0.35克的NaHCO 3，5.96克HEPES至1升pH调至7.35，用NaOH 溶液B:溶液A + 0.04毫谷氨酰胺溶液C:。溶液A + 0.2毫米谷氨酰胺SOLUT离子D:解决方案A + 1毫米谷氨酰胺解决方案E:解决方案A + 5毫米谷氨酰胺溶液F:解决方案A + 5毫米阿拉巴马州

Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

Glutamine Flux Imaging Using Genetically Encoded Sensors

本文将展示如何利用FRET监测谷氨酰胺动态活细胞。遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。实验设计，实验设置的技术细节，并考虑后期的实验分析将用于基因编码的谷氨酰胺的传感器进行讨论。

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 ...

glutamine-flux-imaging-using-genetically-encoded-sensors

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K 次观看.  Virginia Tech. 本文将展示如何利用FRET监测谷氨酰胺动态活细胞。遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。实验设计，实验设置的技术细节，并考虑后期的实验分析将用于基因编码的谷氨酰胺的传感器进行讨论。 

视频: Glutamine Flux Imaging Using Genetically Encoded Sensors

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

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 ...

生物工程

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

利用 nlsgps1 förster 共振能量转移 (fret) 生物传感器可视化细胞赤霉素水平

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

发育生物学

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

神经科学

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

谷氨酰胺通量成像使用基因编码的传感器

摘要

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此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

FIBS功能的无创性代谢轮廓

利用 nlsgps1 förster 共振能量转移 (fret) 生物传感器可视化细胞赤霉素水平

在基于生物发光钙指标GFP的水母体内脑功能成像方法

高通量筛选和生物传感与荧光 C。线虫菌株

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

可筛选的

谷氨酰胺通量成像使用基因编码的传感器

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

FIBS功能的无创性代谢轮廓

利用 nlsgps1 förster 共振能量转移 (fret) 生物传感器可视化细胞赤霉素水平

在基于生物发光钙指标GFP的水母体内脑功能成像方法

高通量筛选和生物传感与荧光 C。线虫菌株

高通量钙流量测定对甘氨酸/d-丝氨酸和谷氨酸敏感的 NMDA 受体的研究

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

可筛选的

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取