The authors acknowledge C.J. Edgell, Pathology Department, University of North Carolina at Chapel Hill, NC, USA for providing the EA.hy926 cells. Author E.E. is supported by Nikon Austria within the Nikon-Center of Excellence, Graz and is a fellow of the Ph.D. program in Molecular Medicine at the Medical University of Graz. The researchers are also supported by the Ph.D. program Metabolic and Cardiovascular Disease (DK-W1226) of the Medical University of Graz. This work was also funded by the FWF project P 28529-B27. Microscopic equipment is part of the Nikon-Center of Excellence, Graz that is supported by the Austrian infrastructure program 2013/2014, Nikon Austria Inc., and BioTechMed, Graz.

1.化学缓冲液和溶液的制备<ol><li>制备含有135 mM氯化钠，5mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，10毫米的HEPES，2.6毫摩尔的NaHCO 3，0.44毫摩尔KH 2 PO 4，0.34毫摩尔的 Na 2 HPO 4，10mM的D-存储缓冲器葡萄糖，2mM的L-谷氨酰胺，1×MEM维生素，1×MEM氨基酸，1％笔链球菌和1％两性霉素B溶解所有组分在蒸馏水中并用磁力搅拌器在室温下搅拌该缓冲器20分钟。调整使用1M的NaOH pH至7.44。滴加氢氧化钠，使用pH计，同时连续搅拌测量pH。 </li><li>制备生理钙含缓冲它由140 mM氯化钠，5mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，10毫D-葡萄糖，和10mM HEPES。如步骤1.1中所述pH调节到使用NaOH 7.4。 </li><li>准备它由相同成分的无Ca 2+缓冲如列在步骤1.2。使用1毫米EGTA，而不是2毫米。 </li><li>溶解的Fura-2AM在DMSO，得到1mM储备溶液。分装在密闭小瓶中在-20℃下长达一个月原液和存储。如果冻结，使储液在室温下平衡避光至少1小时。稀释的Fura-2AM原液在1ml缓冲存储器（步骤1.1），以获得3.3微米的最终浓度。 </li><li>制备1毫升蒸馏水的100mM组胺原液（pH 7.0）中。稀释在100ml 的 Ca 2+的100mM的组胺原液含生理缓冲液至100μM组胺的终浓度。 </li><li>溶解在100毫升的含钙生理缓冲液的Nω硝基-L-精氨酸（L-NNA），得到的300μM的最终浓度。储存在37℃的水浴的解决方案，为至少1小时，直到L-NNA完全溶解。 </li><li>溶解10毫克的NOC-7在蒸馏は之三（pH7.0）中，得到10mM的储备溶液。分装在小等份的原液在密闭小瓶中并在-70℃下立即存储。稀释在生理缓冲液中的NOC-7原液以获得10微米的NOC-7的终浓度。 注:NOC-7是自发释放NO具有短半衰期的小的化学化合物。总是准备，由NOC-7之前，为了每个实验工作缓冲区。 </li><li>使在生理钙缓冲液中的1mM的硝普钠（SNP）的溶液和制备10μM的稀释度。 注:请始终准备少量（约10毫升），因为快速降解率的NO供体的解决方案。 </li><li>制备磷酸盐缓冲液（PBS）组成的137 mM氯化钠，2.7毫摩尔的KCl，9.2毫摩尔的 Na 2 HPO 4，和1.5毫摩尔KH 2 PO 4。调节pH值至7.44用NaOH / HCl中。 </li></ol> 2.细胞的制备注:在邻即刻申请达到均匀使用geNOps测量单个细胞一氧化氮（NO•）的性能，将细胞需要与铁含有之前成像实验缓冲器以恢复的 Fe 2+（II）/维生素C预温育该NO•-probes的NO• - 结合域内。在将EA.hy926和HEK293细胞的情况下，温育20分钟与铁（II）增压溶液导致NO•传感器的完全激活。 <ol><li>种子5.5×10 5个将EA.hy926或HEK293细胞次日或3.5×10 5细胞一天后第二天，在30毫米的显微镜盖玻片到6孔板的孔中。孵育在37℃下细胞在5％的CO 2的潮湿的环境。 注:对于哺乳动物细胞和病毒结构的腺病毒感染的详细的信息已由Zhang 等人所描述。 25。这一步不适用于HEK 293细胞稳定表达G-geNOp。 </li><li>取胎牛血清（FCS）和Dulbecco氏鹰改性培养基（DMEM）无抗生素并添加腺相关病毒5型（AAV5）载体携带的编码为G-geNOp（MOI基因:500为将EA.hy926细胞产率几乎是100 ％阳性细胞; MOI:1是有效的HEK293细胞）。 注:腺相关病毒载体的使用被分配到危险组2生物安全级别2容纳通常需要用于与该载体的工作。如果需要的话，病毒感染，也可以在含有介质FCS的执行。可选择地，细胞可以瞬时使用基于脂质的载体1转染。 </li><li>除去培养基洗细胞用预热的（37℃）的PBS。加入1ml的DMEM / AAV5培养基上每孔1小时。这一步不适用于HEK 293细胞稳定表达G-geNOp。 </li><li>上每孔加入1 ml 20％FCS的含DMEM中以10％FCS的终浓度。不要从细胞中除去含有AAV5媒体。根特LY跷跷板板以均匀的孔中的培养基。孵育细胞48小时，在37℃在5％的CO 2的潮湿的环境。此步骤不适用于在HEK 293细胞中稳定表达的G-geNOp。 </li><li> 48小时后，洗细胞用预热的PBS。接着于室温室温加2ml预热存储缓冲液（第1.1节），以每孔并孵育细胞至少1小时，从光辐射的保护。 </li><li>在室温下以1ml /孔的铁（II）的增压溶液（RT）替换存储缓冲器。孵育细胞在黑暗中恰好20分钟。 注意:不要超过或减少最佳孵育时间，因为这可能会影响NO•探测器的响应。 </li><li>具有存储缓冲液洗涤细胞一次，并以允许细胞平衡用2ml存储缓冲器孵育各孔在RT至少2小时。 </li><li>在RT替换在1ml存储缓冲器与3.3μM的Fura-2AM存储缓冲器45分钟，被保护的从光。 </li><li>具有存储缓冲液洗涤细胞两次并孵育，再次为至少30分钟，以允许细胞达到平衡。 </li></ol> NO•和Ca单个细胞2+信号3.活细胞成像<ol><li>修复涂有金属灌注腔EAhy.926或HEK293细胞（来自步骤2.1）30毫米的盖玻片并将其放置在显微镜。连接涌入管到缓冲水库和外排至真空泵。确保一致的流程，避免盖玻片排水。 </li><li>使用半自动灌注系统开始与生理钙缓冲液（来自步骤1.2）的重力驱动的灌注。 注:这种系统由缓冲水库，各自管道，这是电子控制的磁阀和一个真空泵（ 见图1B）的。的流率范围可以从1至3毫升/分钟，依赖于灌注储层的高度。对于一贯局部药物APPLI阳离子，所有使用的储层的流速应大致相等。这应该是前成像实验测试。认为内皮细胞响应增加其可通过严格灌注来诱导剪切应力。 </li><li>切换成像系统上，并允许预热30分钟的所有设备。 </li><li>定义使用相应的软件成像设置。选择激发波长340 nm和呋喃-2成像380纳米和用于激励的G-geNOp 480纳米。为了最大限度地减少荧光漂白增加相机分级〜4和减少激发强度和暴露时间。参见步骤3.6-3.8。 注:成像设置和参数取决于所使用的设备上，呋喃-2装载效率和G-geNOp表达水平。 </li><li>通过移动显微镜的XYZ表直到几个荧光细胞选择摄像区域是在聚焦。然后定义的通过相应的软件工具利率（投资回报）​​的地区。绘制覆盖整个几个地区SINGL每手动成像电子商务领域的荧光细胞。此外，定义大小相似的背景区域。 注意:一旦图像被获取并存储的ROI，也可为使用各自的成像分析软件进一步分析成像过程后重新定义（参见材料列表）。 </li><li>开始有机动样品台的倒置和先进的荧光显微镜，和单色光源收集数据。分别交替地激发在340nm和的Fura-2AM 380 nm和480nm处为G-geNOp。设置各自的曝光时间，使得所有通道，一个清晰的荧光信号是随时间可检测的。参见步骤3.4。 注意:这取决于激发光的强度和相机分箱。例如，使用的激发光的15％的强度，为4的照相机分级和150毫秒为340纳米，50毫秒为380纳米，300毫秒为480纳米。参见步骤3.4。 </li><li>收集所发出的光在对于呋喃-2 / AM 510纳米，对于G-格诺520纳米p使用电荷耦合器件（CCD）相机组成的500纳米激励器，一个495纳米分色和510-520 nm的发射器的适当滤波器集合。记录中的一个总的框架，每3秒。 </li><li>记录第一分钟（如果必要达3分钟）在生理钙缓冲液（1.2）以上的时间，以获得相应的荧光信号的基线。 </li><li>一旦一个稳定的基线荧光观察，切换到包含生理的 Ca 2+缓冲液来刺激细胞3分钟为100μM组胺或ATP（1μM或100μM）。 注:对激动剂响应将EA.hy926细胞显示的呋喃-2比率（荧光在340nm处通过荧光在380nm处激发除以激发）和G geNOp荧光信号的明确减少急剧增加。 </li><li>切换回生理钙缓冲而不组胺或ATP和L-NNA 5分钟以从细胞除去的化合物。此步骤可能会延长，直至fluores萤光变化完全恢复。 </li><li>管理10μM的NOC-7在生理钙离子的缓冲液使用灌注系统2分钟。 NO供体强烈影响的G-geNOp荧光通常由&gt; 20％的响应于10μM的NOC-7减小。在将EA.hy926细胞相比，诱导G-geNOp荧光猝灭激动剂的NOC-7的影响大约是3倍强。 </li><li>洗出NO• -释放的化合物与生理钙约10分钟2+缓冲和停止记录一次基础荧光恢复。 </li></ol> 4.数据分析<ol><li>出口获取的单个细胞的平均荧光强度数据随着时间的推移，对数据分析软件。 </li><li>减去各自背景值和随时间由每个单细胞的各自的呋喃-2信号380纳米计算的340纳米的比率。 </li><li>减去的G geNOp信道的背景值，以获得实际的FL荧光使用任何计算软件的NO探针（F）随时间的强度。 </li><li>取基线荧光值为F 0（F 0是否探针的荧光随时间无刺激）。参见步骤4.5和图1C。 </li><li>计算的函数随时间使用以下等式荧光漂白效果:F 0 = F inital•EXP（-K•时间）+ F 高原 。 ˚Finital:最大荧光信号，一旦成像启动; K:荧光漂白随着时间的推移的速率常数; ˚F 高原 :最低荧光漂白随着时间的推移达到;参见步骤4.3- 4.4和图1C。 注:对于近似，可能被用来在之前和细胞刺激后的时间全部荧光值。进一步的细节由宾利等规定。 27。 </li><li>随着时间的推移正常化的G geNOp信号，计算1-F / F0（步骤4.3-4.5）。参见图1C和1D。 </li></ol>

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EE，MW，RM和WFG，格拉茨医科大学的工作人员，已经向英国专利申请（专利申请号WO2015EP74877 20151027，优先数GB20140019073 20141027）描述在这个手稿的研究的一部分。与此相关的专利许可提供给下一代荧光成像（NGFI）有限公司（ <a href="http://www.ngfi.eu/" target="_blank">http://www.ngfi.eu/</a> ），分拆公司格拉茨医科大学。

因为NO•的发现如在生物学28的重要信号分子，在单细胞，组织和整个动物的可行和可靠的方式高分辨率游离基的具体实时测量已渴望。在这里，我们报告了最近开发的基因编码荧光NO•探头（geNOps）采用宽视场荧光显微镜1，使精确NO•信号的活细胞成像应用。 规避精细和侵入性的转染程序稳定表达绿色荧光的G-geNOp用于定量外源产生的单细胞否•型材的HEK细胞克隆。如HEK细胞通常不产生NO•内源性29，这种细胞类型是适合于产生一个基于geNOp传感器细胞系的，这可能是在共培养条件的许多其它应用中有用由于没有生产•原代细胞，甚至在活体动物30。然而，在此研究中，我们证明各种否•-liberating化合物的能力，将不同浓度和稳定性的，使用的G-geNOp表达的HEK细胞模型，以唤起胞内NO•信号。我们的数据显示，该NO•端供体的浓度，施用的质量和方法最终确定的细胞内NO•剖面的模式。这种信息对于不同的NO供体•，这是表明几种疾病的原位药代动力学研究是必不可少的。值得注意的是，geNOps已经示出在一个很长的时间1为NO•端供体脉冲的多个重复应用以稳定地作出响应。因此，利用NO•-liberating本文提供的化合物的实验中，允许有关各种幅度和相应的细胞NO的动力学半定量的结论226;信号（ 图1和2）。 虽然稳定表达的HEK细胞克隆可能是从一个单一的细胞起源中，观察到G-geNOps表达水平的一个广泛的异质性（ 图1）。这是稳定的细胞克隆的共同特征为感兴趣的（基因组整合）基因的转录是多种因素的控制下，例如，影响细胞的生长率32多样的环境胁迫31和细胞周期状态33。单基于FP-geNOps是非比例式传感器，因此，使NO•诱导与geNOp表达水平1荧光强度增加的损失。因此，geNOps信号正常化是特别是在比较分析的情况下，量化细胞NO•信号至关重要。如图所示在我们最近的研究中，有严格的线性关系between geNOps的基础荧光强度与NO•诱导荧光猝灭的在宽范围的荧光强度的强度已发现1。这是geNOps用于蜂窝NO•信号的绝对定量的一个重要特征。 如图1中 ，G-geNOps信号的归一化响应于NOC-7和SNP发现均质否•在不同HEK细胞信号从相同的板，这表明HEK细胞不多样与问候其占用容量并降低了NO•自由基是从NO•供体起源。与此相反，在HeLa细胞用geNOps证实蜂窝否•不同小区间的信号的清晰均质响应于NOC-7。这些差异指向细胞类型特异性NO•代谢和分解速率，这可能会在细胞生理和病理多种含义，并且可以使用geNOp被发现的技术。 尽管如此，geNOps的两个重要特性必须要认真考虑的传感器和数据解释的正确用法:I）geNOps需要足够的铁（II），以充分应对NO•1取决于FP变型II），geNOps可以是pH敏感1。在这里，我们描述了已被发现是适合于geNOps，其在任一HEK，海拉或将EA.hy926细胞（参见协议2.6）表示的无毒铁（II）补充的协议。虽然已经证明，与铁细胞处理（Ⅱ）/维生素C不影响细胞形态，细胞活力和细胞1的代谢活性，这可能是必要的，以优化其他细胞类型和组织这一重要步骤。然而，在某些实验条件下，铁（II）负载的要求可能会限制geNOps的适用性。值得注意的是，它已经显示，抗坏血酸可以减少氮素O•35和抗坏血酸-铁（Ⅱ）配合物是能够清除NO•36,37。此外，过量的铁（II）和抗坏血酸可诱导的炎症反应39和不耦合的eNOS 41。这种影响需要使用geNOps技术时，必须考虑。已经显示，在某些实验条件下，细胞内pH值的影响显著34，其具有以影响geNOps荧光1的潜力。值得注意的是，青色和绿色geNOps变体是相对pH敏感表示荧光的酸化时1的降低。因此，（亚）细胞pH为急性变化可能使用pH敏感geNOps当模拟假否•信号。所建议的前期工作，NO•不敏感geNOps并行使用（geNOps MUT）为阴性CONTROLS建议从pH值的变化1解剖真正的蜂窝NO•信号。除了蜂窝pH变化可使用pH探头如SypHer 34进行检查。 此外，我们在响应于内皮细胞替代将EA.hy926生理钙 -mobilizing激动剂可视内源性酶否•形成。在将EA.hy926细胞系一贯表达的eNOS 38经常使用的模型系统。在将EA.hy926细胞瞬时表达geNOps的使用，确认该IP 3介导的 Ca 2+信号唤起深刻否•形成在此细胞类型，这是几乎完全由L-NNA阻断。为了与否•信号在时间上相关的 Ca 2+，G-geNOp表达细胞装载有紫外激发的化学钙 -indicator呋喃-2 / AM。 CA的光谱分离2+绑定和非绑定呋喃-2 FLUO<html>从G-geNOp信号rescence可以用市售滤波器设置40很容易地实现。成像两种探针推出的钙 -triggered酶NO•形成对比胞质钙离子在这种内皮细胞类型出现上升慢得多。在细胞处理的单细胞否•在从牛肺动脉血管内皮细胞的信号的类似动力学与IP 3 -产生激动剂缓激肽，以及剪切应力已使用报道NOA-1，一种间接的高度否•敏感的传感器12 。因此，这些数据强调， 钙 -evoked的eNOS衍生的NO•形成需要一定的起动时间，直至达到充分的酶活性。虽然蜂窝否•阵型，扩散和降解的动力学可以从其它数据中提取， 例如，基于张力-NO•测量诱导的血管舒张SS ="外部参照"&gt; 26，NO荧光探针•的巨大好处是，他们直接将蜂窝NO•波动的实时可视信号。因此，成像细胞NO•与geNOps信号提供了高时空分辨率，并在（再）提供了独特的可能性调查（子）蜂窝NO•动态平衡。例如，成像的eNOS与geNOps技术在单个内皮细胞组合的穿梭42可能是合适的关联否•形成与NO•的亚细胞定位和易位-producing酶或其他相关的蛋白质如钙调蛋白和小窝蛋白43。 这里，我们描述的G-geNOp的表达的HEK和将EA.hy926细胞可视化外生和内源性生成的蜂窝否•信号在单细胞水平和实时上的传统的宽视场荧光微米切实可行应用icroscope。我们的数据暗示geNOps适合于具体跟踪使用各种有趣的细胞类型的各种实验条件下（子）蜂窝否•动力学。

我们最近开发一类新的遗传编码的荧光否•探针，称为geNOps 1。这些传感器包括一个简单地建造，细菌衍生的，编号•结合结构域2，其缀合于不同的荧光蛋白（FP）变体3（青色，绿色或橙色，FP）的。 NO•在geNOps内绑定到非血红素铁（II）中心4即刻降低了荧光强度1。重要的是，当细胞内NO•水平下降1 geNOps荧光迅速全面复苏。因此，geNOps允许（子）蜂窝NO•波动实时成像。虽然NO•传感geNOps的机制仍不清楚，到目前为止，它们已被证明是优良的NO•记者，因而有效力开拓的多色的，定量NO•bioimagin一个新的时代克具有高空间分辨率和时间分辨率1,5。其他可用的荧光否•探针基于小化学化合物，其需要被加载到细胞中，并通过否•6不可逆地修改。 NO•敏感荧光小的其它缺点是其潜在的细胞毒性和相对较低的特殊性，这使得它难以可靠，分析和确凿的方式7，8，9使用它们。虽然基因编码荧光探针的有效使用，需要高效的基因转移技术，基于FP遗传编码传感器已成为已彻底改变了我们的细胞10，11的内运作的理解不可或缺的工具。单基于FP-geNOps的发展，一架F之前örster共振能量转移（FRET）为主否•传感器称为NOA-1 12构建。 Sato 等人。设计这个复杂探测器由NO•敏感的可溶性鸟苷酸环化酶的两个亚基（SGC），两者结合到基于FRET的传感器报告的环磷酸鸟苷（cGMP）的水平12。由于这种探测器响应cGMP的，它只是间接地检测细胞内NO•波动12。虽然NOA-1响应为NO•在纳米摩尔范围立面图，此工具尚未频繁使用到目前为止，可能是由于与本笨重偶传感器的可用性和实用性的限制。 否•，这影响基本生物过程已被充分表征13,14的多种功能。许多研究证明，NO•concentratioN个单元和子域范围内决定健康和疾病的14，15，16细胞的命运。在哺乳动物，编号•主要由良好表征的一氧化氮合酶（NOS）家族17产生酶在各种细胞类型。到目前为止，NOS的三种亚型已经描述18，19，20;这些是的Ca 2+ /钙调蛋白依赖性内皮型NOS（eNOS的或NOS-3）18和神经元型NOS（nNOS的或NOS-1）19和钙 /钙调蛋白依赖性组成型活性诱导型NOS（iNOS的或NOS- 2）20。此外，线粒体NOS（mtNOS）的存在也被建议21。然而，mtNOS被认为的nNOS的剪接变体，并且因此不单独分类<html>作为一个亚型21。另一个同种型，除了那些在哺乳动物细胞，是所谓的细菌NOS（BNOS），主要发现在革兰氏阳性菌22。酶产生NO•的高度控制，并取决于几个辅因子的可用性，例如烟酰胺腺嘌呤二核苷酸磷酸（NADPH），黄素腺嘌呤二核苷酸（FAD），四氢（BH 4），分子氧和L-精氨酸17。阳离子氨基酸L-精氨酸]是在否•生产17转化为L-瓜氨酸的衬底。除了高度管制酶促生成NO•的，它已被假定的是基团可非酶由线粒体亚硝酸盐池低氧条件23减压。一旦否•是细胞内产生的，它可以自由地通过生物膜14扩散，裁判"&gt; 15，但是，这种激进的很短的半衰期，主要由环境条件决定，和各种途径和化学反应有效地降解NO•级别24，最终，编号•的形成，扩散和降解取决于上不同的环境参数而确定的高度生物活性分子24的有效浓度。 所述geNOps技术允许直接检测的（亚）细胞否•波动1，因此是合适的重新调查，和新发现负责蜂窝NO•信号的累积和分解的机制。在这里，我们提供简单的协议和geNOps的使用形象化外生诱发代表性结果和内源性生成的NO•型材，对单个细胞的水平。此外，该geNOps技术可适于AP折襞在其它细胞模型系统以研究响应于不同的细胞刺激和应力NO•形成，扩散和降解的复杂的图案。

<table><tbody><tr><td>NaCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>3957.20</td><td>sodium chloride</td></tr><tr><td>KCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>6781.1</td><td>potassium chloride</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>T885.1</td><td>calcium chloride dihydrate</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>2189.2</td><td>magnesium chloride hexahydrate</td></tr><tr><td>HEPES</td><td>Carl Roth, Karlsruhe, Germany</td><td>9105.3</td><td>4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Carl Roth, Karlsruhe, Germany</td><td>8551.1</td><td>sodium hydrogencarbonate</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck, Darmstadt, Germany</td><td>104873</td><td>potassium dihydrogen phosphate</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth, Karlsruhe, Germany</td><td>4984.1</td><td>sodium hydrogenphosphate dihydrate</td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Carl Roth, Karlsruhe, Germany</td><td>6780.1</td><td>&gt;99.5%; for cell culture, endotoxin free</td></tr><tr><td>EGTA</td><td>Carl Roth, Karlsruhe, Germany</td><td>3054.2</td><td>ethylene glycol-bis(2-aminoethylether)-&lt;em&gt;N&amp;#8288;,&amp;#8288;N&amp;#8288;,&amp;#8288;N&amp;#8288;&amp;prime;&amp;#8288;,&amp;#8288;N&amp;#8288;&amp;prime;&amp;#8288;&lt;/em&gt;-tetraacetic acid; calcium chelating agent</td></tr><tr><td>NaOH</td><td>Carl Roth, Karlsruhe, Germany</td><td>6771.1</td><td>sodium hydroxide</td></tr><tr><td>HCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>4625.1</td><td>hydrochloric acid, fuming, 37% (~10 N)</td></tr><tr><td>DMSO</td><td>Carl Roth, Karlsruhe, Germany</td><td>4720.1</td><td>dimethyl sulfoxide;&amp;nbsp; highly polar, aprotic organic solvent</td></tr><tr><td>L-Glutamic acid hydrochloride&amp;nbsp;</td><td>Sigma Aldrich, Vienna, Austria</td><td>G2128</td><td>(S)-2-Aminoglutaric acid</td></tr><tr><td>DMEM, low glucose</td><td>Sigma Aldrich, Vienna, Austria</td><td>D5523</td><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - low glucose; with 1,000 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture&amp;nbsp;</td></tr><tr><td>MEM Vitamin solution (100x)</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>11120037</td><td>100x the vitamins found in the standard Minimum Essential Medium (MEM)</td></tr><tr><td>MEM Amino acids solution (50x)</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>11130036</td><td>50x the essential amino acids (except L-glutamine) found in the standard Minimum Essential Medium (MEM)</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>15140122.00</td><td>antibiotics to prevent bacterial contamination of cell cultures</td></tr><tr><td>Amphotericin B</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>15290026.00</td><td>Gibco Amphotericin B contains 250 &amp;micro;g of amphotericin B (Fungizone) and 205 &amp;micro;g of sodium deoxycholate; prevents the contamination of cell cultures by yeast and multicellular fungi</td></tr><tr><td>FCS</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>10270106</td><td>Fetal Bovine Serum, qualified, E.U.-approved, South America origin</td></tr><tr><td>PBS, pH 7.4</td><td>Gibco/Thermo Fisher Scientific, Waltham, MA, USA</td><td>10010031.00</td><td>phosphate-buffered saline</td></tr><tr><td>Fura-2 (AM)</td><td>Teflabs, Austin, TX, USA</td><td>102</td><td>fluorescent cytosolic calcium indicators</td></tr><tr><td>Histamine dihydrochlorid</td><td>Sigma Aldrich, Vienna, Austria</td><td>H7250</td><td>2-(4-Imidazolyl)ethylamine dihydrochloride; IP3-generating agonist&amp;nbsp;</td></tr><tr><td>N&amp;omega;-Nitro-L-arginine</td><td>Sigma Aldrich, Vienna, Austria</td><td>N5501</td><td>N5-(Nitroamidino)-L-2,5-diaminopentanoic acid; L-NNA;&amp;nbsp; inhibitor of nitric oxide synthase</td></tr><tr><td>NOC-7</td><td>Calbiochem/Merck, Darmstadt, Germany</td><td>487952</td><td>3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine; nitric oxide (NO) donor short half-life of NO release</td></tr><tr><td>Sodium nitroprusside dihydrate</td><td>Santa Cruz Biotechnology, Texas, USA</td><td>sc-203395A</td><td>sodium nitroferricyanide(III) dihydrate; nitric oxide releasing compound</td></tr><tr><td>30 mm Cover slips</td><td>Karl Hecht, Sondheim v. d. Rh&amp;ouml;n, Germany</td><td>41001130</td><td>glass cover slips, HECHT &quot;Assistent&quot;, size 1, round, 30 mm, (VE: 100 pcs.)&amp;nbsp;</td></tr><tr><td>Iron(II) booster solution</td><td>Next generation fluorescent imaging (NGFI), Graz, Austria</td><td>n.a.</td><td>Iron(II) containing physiological buffer for non toxic iron(II) loading of cells;&amp;nbsp; http://www.ngfi.eu/product/ironii-booster-solution/</td></tr><tr><td>G-geNOp plasmid</td><td>Next generation fluorescent imaging (NGFI), Graz, Austria</td><td>n.a.</td><td>plasmid DNA encoding green&amp;nbsp;NO sensitive probe (G-geNOp);&amp;nbsp; http://www.ngfi.eu/product/g-genop/</td></tr><tr><td>G-geNOp AAV5</td><td>Next generation fluorescent imaging (NGFI), Graz, Austria</td><td>n.a.</td><td>adenovirus encoding green NO sensitive probe (G-geNOp);&amp;nbsp; http://www.ngfi.eu/product-category/genops/viral-genops-vectors/g-genop-aav5/</td></tr><tr><td>G-geNOp sensor cell line</td><td>Next generation fluorescent imaging (NGFI), Graz, Austria</td><td>n.a.</td><td>human embryonic kidney cell line (HEK293) stably expressing green NO sensitive probe (G-geNOp);&amp;nbsp; http://www.ngfi.eu/product-category/genops/g-genop-sensor-cell-line/</td></tr><tr><td>HEK293A cell line</td><td>Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA</td><td>R70507</td><td>subclone of human embryonic kidney cell line (HEK293)&amp;nbsp;</td></tr><tr><td>EA.hy926 cell line</td><td>American Type Culture Collection (ATCC), Wesel, Germany</td><td>CRL-2922</td><td>somatic cell hybrid clone of human umbilical vein cell line with a thioguanine-resistant clone of A549</td></tr><tr><td>TILL iMIC</td><td>Till Photonics, Graefling, Germany</td><td>n.a.</td><td>digital microscope</td></tr><tr><td>Polychrome V monochromator</td><td>Till Photonics, Graefling, Germany</td><td>n.a.</td><td>ultra fast switching</td></tr><tr><td>AVT Stingray F145B</td><td>Allied Vision Technologies, Stadtroda, Germany</td><td>n.a.</td><td>Versatile CCD camera with Sony ICX285 EXview HAD sensor, IEEE 1394b</td></tr><tr><td>alpha Plan Fluar 40</td><td>Zeiss, G&amp;ouml;ttingen, Germany</td><td>n.a.</td><td>40X objective</td></tr><tr><td>dichroic filters</td><td>Chroma Technology Corp, Rockingham, Vermont, USA</td><td>n.a.</td><td>GFP emitter 514/3 nm (515dcxr)</td></tr><tr><td>ValveBank8 Controller</td><td>AutoMate Scientific, Inc., Berkeley, California, USA</td><td>01-08</td><td>programmable perfusion system control unit</td></tr><tr><td>BVC control</td><td>Vacuubrand, Wertheim, Germany</td><td>727200</td><td>Chemistry diaphragm pump ME 1C; vacuum pump for perfusion system</td></tr><tr><td>ImageJ software</td><td>&amp;nbsp;NIH Image&amp;nbsp;</td><td></td><td>Java image processing program inspired by NIH Image. http://imagej.net/Welcome</td></tr></tbody></table>

application of genetically encoded fluorescent nitric oxide (no&#8226;) probes, the genops, for real-time imaging of no&#8226; signals in single cells

一氧化氮（NO•）是一个小的基团，其介导在哺乳动物，细菌和植物的多个重要的细胞功能。尽管有大量的用于在体内和体外检测NO•方法的存在，在单细胞水平NO•的实时监控是很有挑战性的。 NO•的生理或病理作用是通过实际的浓度决定和住这种激进的时候。因此，允许NO•的单细胞的检测方法是非常需要的。最近，我们通过引入单个荧光蛋白质基遗传编码一氧化氮（NO•）探针（geNOps），该直接响应蜂窝NO•波动，因此，解决了这一需求扩大的否•指标托盘。这里，我们证明geNOps的使用情况，以评估应对两个不同的化学NO•-liberating分子细胞内NO•信号。我们的研究结果阿尔斯如与比较ö证实，新鲜制备的3-（2-羟基-1-甲基-2-亚硝基肼基）-N-甲基-1-丙胺（NOC-7）具有高得多的电势，以唤起在细胞内NO•水平变化无机NO供体•硝普钠（SNP）。此外，进行使用绿色geNOps（G-geNOp）和化学的 Ca 2+指示剂呋喃-2双色活细胞成像可视化钙的紧调节依赖性否•形成在单一的内皮细胞。这些代表性实验证明geNOps是调查在不同的实验设置单细胞NO•信号的实时生成和降解合适的工具。

为了应对瞬时应用NO供体单细胞NO•型材可视化我们使用的HEK细胞克隆稳定地表达的G-geNOp（ 图1A）中，为了可视化在单细胞水平否•信号响应于两个不同的NO-释放小的化学化合物，NOC-7和SNP。使NO•供体使用，确保连续流动（ 图1B）基于重力灌注系统成像过程中连续施加到并从细胞中除去。表达的G-geNOp具有不同强度的所有细胞显示出响应的明确减少荧光的至NOC-7和SNP（ 图1C），这表明在加入的NO•供体的细胞内的快速否•积累。标准化荧光信号（1-F / F 0）表明，这两种否•供体诱发均匀cellulaR无•经NO• -释放的化合物（ 图1D）的冲洗是完全恢复高程。然而，10μM的SNP诱导仅50％的蜂窝NO•信号（9.63±1.05％，N = 3/38），这是由10微米的NOC-7（18.10±1.20％，N = 3/38，对达到&lt; 0.0001）。为了达到相等的细胞内NO•水平与两个否•供体，被要求的SNP为1mM的浓度（ 图1C，1D）。 下一步，我们测试制备与过期的NOC-7在HEK细胞升高的细胞内NO•水平的新鲜的能力。为了这个目的，我们准备了含有5μM的NOC-7四个实验缓冲器。使NO•供体要么测量新鲜刚刚之前加入，或保持油藏内为1小时，2小时，并在室温下测量之前3小时。在不同的缓冲器中连续施加及除去˚FROM使用灌注系统（ 图2B）的G geNOp表达细胞。这种方法推出含水的NOC-7溶液，其如预期显示出减少随时间升高的细胞内NO•水平（ 图2A，2C）的能力的稳定性。有趣的是，编号•信号显著更快一旦去除过期缓冲器的回收相比，这是由新鲜的NOC-7（ 图2C），在细胞成分的完整否•-liberating分子也许指示粘附诱发的细胞内NO•响应。 钙的同时可视化和NO•单内皮细胞信号为了研究钙在血管内皮细胞-triggered NO•形成，常用的内皮细胞永生化，替代，将EA.hy926细胞系，呈丁使用G-geNOp ransiently转染并载带fura-2 / AM（见协议2.8）。转染得到的G-geNOp阳性内皮细胞的大约10％（N = 6， 图3A），这是足以记录的 Ca 2+ -evoked单内皮细胞的NO水平•生产。但是，我们取得了使用腺相关病毒载体近100％的G-geNOp阳性细胞将EA.hy926（N = 6;见协议2.2）。前多通道测量，将细胞在储存缓冲液中孵育20分钟，在室温下由L-NNA，一种有效的不可逆的NOS抑制剂26。控制细胞保持在同一个存储缓冲器没有L-NNA（见协议1.1）（ 图3B）。对照细胞组胺是一种有效的1,4,5-三磷酸（IP 3） -产生激动剂，瞬间提升细胞内钙水平后细胞内NO•逐渐增加，直到激动剂是删除处理D（ 图3C，3D）。细胞用的NOS抑制剂预处理表现出相似的细胞内的Ca 2+信号，而细胞内NO•水平仍响应于组胺（ 图3E）几乎不受影响。将EA.hy926细胞中表达的G-geNOp也用1μM处理和100 -产生激动剂ATP为了测试geNOps是否是合适的，以监测响应否•信号都低生理和超生理的IP 3微米激动剂（ 图3F）的浓度。在内皮细胞株为1μMATP诱发的明确胞质否•信号，为约100μMATP（ 图3F）得到的信号的一半。 <img alt="图1" src="/files/ftp_upload/55486/55486fig1.jpg" /> 图1:针对不同的NO-liberati细胞内NO型材<html>纳克分子。 HEK细胞（A）宽视场图像稳定表达胞质G-geNOp。比例尺= 20微米。 （B）中为NOC-7和SNP的控制施加和去除基于重力的半自动灌注系统的示意图。 （C）的代表性的（在3次独立实验）非归一化的单细胞荧光强度以任意单位迹线与HEK细胞稳定表达胞质的G-geNOp响应于10μM的NOC-7，10μM的SNP的时间，和1mM SNP。粗黑的曲线代表的26个单细胞的痕迹平均曲线（浅灰色曲线）。点缀黑色曲线表示F 0，它被用于归一化。 （D）的归一化和反向单迹线（1-F / F 0，浅灰色曲线），和平均曲线（黑色粗体曲线）随时间响应于10μM的NOC-7，10μM的SNP，和1mM的SNP从提取面板C.ES / ftp_upload / 55486 / 55486fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55486/55486fig2.jpg" /> 图2:采用稳定的G-geNOp表达HEK细胞NOC-7的稳定性试验。 （A）代表NO•根据以上的新老NOC-7的缓冲溶液应用表达HEK细胞稳定G-GeNOps时间浓度响应曲线。用使用相同的储备溶液（50毫摩尔）5μM的终浓度最初制备所有含有NOC-7缓冲区。制备后的各溶液的经过时间，直到成像达1小时，2小时和3小时所指示的。 （B）基于重力的半自动灌注系统的示意图，连续添加和成像过程中去除含NOC-7的解决方案。 （C）的时空变化细胞NO•响应信号，以新鲜烹制的老NOC-7缓冲区。 <a href="http://ecsource.jove.com/files/ftp_upload/55486/55486fig2large.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/55486/55486fig3.jpg" /> 图3:NO•和钙离子信号在单细胞将EA.hy926同时多通道成像。 （A）的被载带fura-2 / AM（中间和右面板）表达G-geNOp（左图）EAhy.926细胞的代表性宽视场荧光图像。比例尺= 20微米。 （B）的测量之前的6孔板内的治疗EAhy.926细胞与500μM的Nω硝基-L-精氨酸（L-NNA）对于20分钟的示意图。 （C）呋喃-2（激励的同时记录代表时程:340纳米/ 380纳米EMISS离子:510毫微米）和G-geNOp信号（激发:480纳米;发射:响应至100μM组胺520纳米）。正如所指出的组胺用灌注系统3分钟后取出。 （D）的曲线代表细胞内的Ca的同时进行的录像2+（呋喃-2比率信号，女340 /˚F380是灰色实线）和NO•（归一化和倒曲线，1-F / F 0是绿色实线）的信号在一个单一的呋喃-2 /时间我装内皮细胞瞬时表达G-geNOp。细胞用100μM的组胺刺激含缓冲器3分钟的钙离子 （2毫米氯化钙2）（N = 8/3）。 （E）代表同时记录的 Ca 2+（灰色实线）和NO•（绿色实线）的信号通过一个将EA.hy926细胞的时间用500微米Nω硝基-L-精氨酸（L-NNA）预处理前测量（N = 12/3）。细胞在2mM的存在下与100μM的组胺处理钙离子 。 （F）的代表胞质否•响应于1μMATP，接着用100μM的三磷酸腺苷的第二细胞刺激信号的平均曲线。条代表响应于1μMATP（白条）和4个独立实验100μM的三磷酸腺苷（绿色棒）平均值±最大的G-geNOp信号SD; P &lt;0.001对1μMATP。 P值使用不成对t检验计算。 <a href="http://ecsource.jove.com/files/ftp_upload/55486/55486fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Application of Genetically Encoded Fluorescent Nitric Oxide NOâ€¢ Probes, the geNOps, for Real-time Imaging of NOâ€¢ Signals in Single Cells at JoVE.c

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

application-genetically-encoded-fluorescent-nitric-oxide-no-probes

Nanyang Technological University

Research

JoVE Journal

Biology

12.8K 次观看.  Medical University of Graz. This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO•) probes (geNOps) to monitor single cell NO• fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO• formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

视频: Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

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

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遗传学

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

免疫与感染

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 ...

生物工程

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

发育生物学

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

构建基于细胞的神经递质荧光工程记者（CNiFERs）神经递质的光学检测<em&gt;在体内</em

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

神经科学

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 ...

生物学

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

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