Name: use of enzymatic biosensors to quantify endogenous atp or h2o2 in the kidney
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Description: Watch this Scientific Journal Video about Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney at JoVE.com

我们赞赏Sarissa的生物医学为他们的发展在目前的手稿所使用的传感器的工作。这项研究是由美国国家心肺支持和血液研究所授予HL108880（A. Staruschenko），HL 116264（A.考利）和HL 122662（A. Staruschenko和A.考利），由医学院资助的一个项目威斯康星州研究事务委员会＃9306830（O. Palygin），并推进一个健康的威斯康星州的研究和教育项目＃9520217，和全国肾脏基金会的青年研究者格兰特（O. Palygin）。

以下动物的程序粘在美国国立卫生研究院指南实验动物的护理和使用。在此之前批准从机构动物护理和使用委员会（IACUC）获得。 注:评论的传感器制造商的说明应在试验设计和其使用前可完成。遵守这些指示使用传感器时，产生最佳的效果。 1.传感器校准<ol><li>制备新鲜储备溶液事先实验开始。 </li><li>创建缓冲液A含有10mM NAPI缓冲液，100毫摩尔NaCl，1mM的MgCl 2的，和2mM甘油。用NaOH将pH调节至7.4。保存在2-8℃。 </li><li>使用库存ATP浓度（100毫米）储存在-20℃下，加入10微升的库存到90微升缓冲液A的创建一个新的10毫摩尔ATP校准溶液</li><li>通过将其前端（图2）进入吨再水合的ATP传感器他补液室含有缓冲液A至少10分钟，在2-8℃。 注:补液后，传感器不应该暴露于空气中超过20秒或传感器灵敏度可降低。如果长时间曝光到空气预计，简要地浸传感器成甘油的溶液中。该传感器可用于多种实验，但这些都必须发生在同一天的传感器补液。该传感器可以被存储在补液腔室的缓冲液A达24小时。 </li><li>打开双通道电位（图3），并开始记录系统软件。 </li><li>制备一个校准室用3ml缓冲液A.将参考电极插入校准室。以每个传感器的补液室，然后将其连接到机械手，将其插入校准室解决方案。 注:使用标准有机硅涂层的培养皿作为校准室。进行所有的校准和ST在上一个高性能的实验室空气表法拉第笼udies在电流分析法记录（ 图4），以减少信号噪声。校准最好做得尽可能靠近数据收集尽可能的开始。 对于体内应用校准的最佳时间是在动物手术后的恢复期。 </li><li> 体外校准<ol><li>通过骑自行车从-500 mV的传感器到+500毫伏，在10个循环的速度100毫伏/秒的执行校准室循环伏安法。这极大地提高了传感器的灵敏度。 参见图5，用于从10个循环中观察到的痕迹。 </li><li>最后一个循环之后极化传感器600毫伏。该传感器电流将衰减到一个渐近线。一个稳定的读数至少5分钟后取得。记录的零读数。 </li></ol></li><li> 体内传感器校准<ol><li>不要在体内的Senso进行循环伏安法RS。而是，偏振在校准室中的传感器30秒〜500毫伏。然后稳压器设置为0毫伏，并允许传感器电流上升到一个渐近线。传感器电流将至少需要2分钟，以渐近线。记录的零读数。 </li></ol></li><li>连续添加ATP的解集量进入腔室，以产生校准线包围一期望的检测范围。的ATP溶液中产生的传感器信号最初的尖峰，随后是衰减的ATP的扩散均匀分布在整个所述腔室。记录信号值一旦信号电平的每个ATP加入后已稳定。 图6A和图7示迹线和建议的ATP浓度为体外和体内研究，分别。 注意:它通过使用分析物的清除剂，以确认在​​电极的选择性是很重要的。本协议所使用apyrase测试ATP传感器和过氧化氢酶对H &lt;的特殊性子&gt; 2 O 2的信号（图6B）。如果药物是待施用，它们的反应性与传感器测量应当在研究前确定。 </li><li>加入3微升apyrase从2毫克/毫升（89 UN /毫克）一个股票以测试ATP传感器（电流通过ATP的应用产生的应减少到零电平（图7）的特异性。 </li><li>加入3微升过氧化氢 ​​酶的从为2mg / ml的库存（100 UN /毫克）检验零传感器的特异性（当前用H产生2 O 2的应用程序应该减少到零读数）。 </li></ol> 2.动物手术传感器研究<ol><li>手术体外 <ol><li>麻醉实验动物用异氟烷（5％诱导，1.5至2.5％维护）/医用级O 2或其他批准的方法。15&#39;12动物必须不断监控，以确保麻醉适当水平。圣能够呼吸频率和脚趾捏的反应是用来确定适当的麻醉。 注:安乐死的动物不按照批准IACUC协议。在完成所有非生存的过程安乐死深度麻醉动物用开胸诱导气胸，以确保动物 12的人道消亡。 </li><li>放置在一个温度控制的手术台大鼠在仰卧位置。同时保持了适当的麻醉深度，使得大约5厘米的正中切口与左肾线和暴露远侧腹主动脉。 </li><li>环绕腹腔及肠系膜上动脉和腹主动脉上述这些动脉连字，但不结扎。裹2连字周围低于肾动脉的腹主动脉。 </li><li>夹紧腹主动脉上面的连字。扎下结扎。导尿腹主动脉用聚乙烯管（PE50）。固定导管与第二主动脉结扎。 </li><li>取出钳和结扎肠系膜和腹腔动脉。灌注到肾脏以6ml /分钟的Hanks平衡盐溶液在RT 2-3分钟，直到肾脏完全变白。 </li><li>切除肾脏和导管，连接在主动脉的部分。将肾成3 ml的培养皿充满浴溶液。 注意:实验协议可以在室温下进行。浴溶液含有以mM:145氯化钠，氯化钾4.5，2的MgCl 2，1的CaCl 2，10 HEPES，pH值7.35，用NaOH调节。 </li></ol></li><li>手术在体内<ol><li>使用麻醉IACUC批准协议的老鼠。 对于体内分析麻醉大鼠用氯胺酮（20mg / kg的即时通讯 ）及inactin（50毫克/千克IP）。动物必须不断监控，以确保麻醉适当水平。稳定的呼吸频率和脚趾捏的反应是用来确定适当的麻醉。 </li><li>经过适当的麻醉深度是获得版，将大鼠在位于空气台温度控制的接地表面仰卧位置。表面应预热并保持在36℃。 </li><li>同时保持适当的麻醉深度，使中线切开约5cm与肾细胞系。 </li><li>用缝合线偏转和锚定皮肤和皮下组织，以便整个肾脏是可见的。将肾脏的肾杯，以尽量减少运动伪影。 </li><li>通过颈静脉的0.9％NaCl在1毫升/ 100克/小时，保持血液体积:使用2％BSA的静脉滴注。导管插入双侧输尿管尿液收集。周围肠系膜和腹腔动脉地点关系和远端主动脉肾灌注压（ 图10A）的操作。 </li><li>如果在体内实验是必需的药剂中的应用，插入一个填隙导管的建议（图10B </strong&gt;）。 </li></ol></li></ol> 3.数据采集设置<ol><li>打开该数据采集程序和为体外和体内实验，以阳极的正设置其极性。设置程序将数据保存为ASCII码。 </li><li>放置显微操作的传感器进入肾脏的快速插入。 注:另外，使用连接到微操作机器人的虚拟探头，以帮助实现传感器所需的位置。 </li><li> 体外数据采集<ol><li>灌注经由插管主动脉浴溶液（来自2.1.6）的肾脏，以650微升/分钟的恒定速度。用手术剪刀，小心地取出肾囊，这是必要的传感器插入。 </li><li>确保与绑在肾脏和连接到硅氧烷涂覆菜销橡皮筋肾。 </li><li>放置参考电极靠近肾脏在培养皿其前端浸没在缓冲溶液。 </li></ol></li><li> 体内的数据采集<ol><li>发生在肾杯肾。根据动物的品系和年龄使用杯保存肾脏松散的尺寸。 图8示出了两种尺寸肾杯。类似杯被用于不同的生理途径集中在肾功能的分析，例如微穿刺等16。 注意:肾脏杯中的位置是至关重要的，以消除该动物呼吸产生的机械噪音，但不应该干扰或阻断肾灌注或尿流。 </li><li>在进行数据收集之前允许的恢复时间45分钟。 </li><li>使用26-30G针，使穿刺孔在肾脏传感器的所需的位置和深度。吸干表面的孔，除去渗出血液。添加甘油溶液到肾脏的表面上。这将防止肾脏表面从在实验期间变干。 </li&gt; <li>从补液室中取出的第一传感器和它连接到微操作机器人。很快，在20秒内，将电极插入肾脏中新创建的孔。重复步骤3.5-3.9为空传感器。 </li><li>插入参比电极进入肾脏，大约1cm从来自传感器。 </li></ol></li><li>打开电位和激活的计算机上记录程序。 图9示出了离体肾脏的数据采集的最后设置。 图10显示了 体内肾数据采集与插入的导管的最终设置。 </li></ol> 4.数据分析<ol><li>导入ASCII数据文件到地或任何其他类似的软件。 </li><li>集中当前关系<ol><li>使用线性拟合/外推功能，建立一个线性浓度（X轴 ），以目前的ATP或H 2（Y轴）的关系</suB&gt; O 2的校准点（图6和7）。 线性拟合线:Y = mx + b中</li></ol></li><li>等同于集中当前</li><li>减去空传感器的测得迹线从这些ATP的传感器以获得由细胞内ATP产生的实际电流。 </li><li>转换使用4.2.1详细校正公式由安培法获得纳米帕ATP值。通过导入各调整电流进入的"x"值并求解Y（分析物的浓度）测定ATP的传感器的减去数据轨迹的浓度。 </li><li>同样地，如果需要的话，从计算其校准方程的 H 2 O 2的浓度。 </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+overflow+in+skeletal+muscle+1A+arterioles.">ATP overflow in skeletal muscle 1A arterioles.</a> J. Physiol. 588, 3089-3100 (2010).</li><li>Nishiyama, A., Jackson, K. E., Majid, D. S., Rahman, M., Navar, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+interstitial+fluid+ATP+responses+to+arterial+pressure+and+tubuloglomerular+feedback+activation+during+calcium+channel+blockade.">Renal interstitial fluid ATP responses to arterial pressure and tubuloglomerular feedback activation during calcium channel blockade.</a> Am J Physiol Heart Circ Physiol. 290, H772-H777 (2006).</li><li>Jin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+renal+perfusion+pressure+on+renal+medullary+hydrogen+peroxide+and+nitric+oxide+production.">Effects of renal perfusion pressure on renal medullary hydrogen peroxide and nitric oxide production.</a> Hypertension. 53, 1048-1053 (2009).</li><li>Khan, A. S., Michael, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+consequences+of+using+micro-electrodes+and+microdialysis+probes+in+the+brain.">Invasive consequences of using micro-electrodes and microdialysis probes in the brain.</a> Trends Anal. Chem. 22, 503-508 (2003).</li></ol>

通过Sarissa的生物医药有限公司（考文垂，英国）的传感器提供该手稿的视频录制。

本协议被开发，以提供增强的ATP的时间和空间分辨率和 H 2 O 2的信令用于离体分离的，灌注和体内血液灌注肾脏。的协议和此处所使用的传感器之间的差异提供最佳的数据采集或者药理学试剂或生理研究。该协议包括1）传感器校准，2）的外科手术，3）数据采集设置，以及4）数据分析。它们使分析物的众多的实验条件的实时测量。这将导致更大的见解肾脏疾病和更有效的药物治疗的开发。在这里，我们使用的传感器来分析ATP和H 2 O的2。然而，传感器的其它物质也可并可以使用。施加此处描述的方案来研究ATP和H 2 O的2在大鼠肾脏，但相同的实现方法具D能很容易地在小鼠中的应用进行调整。因此，这种方法具有很大的潜力考虑遗传啮齿动物模型的丰度。 酶生物传感器微电极的基本设计是在1960年由克拉克和里昂2开发。随着生物传感器的初步发展，取得了进展在设计17-19和应用程序20。 Llaudet 等人开发的21,22，而使用从Cosneir 等人 23的方法的酶的涂层在本协议中使用ATP的传感器的原理设计。这些传感器检测到的ATP信令在许多生理过程，包括ATP释放从脑星形胶质8，呼吸4,24的调节，以及骨骼肌动脉第 25条。最近的体外协议已被用来测量ATP的信令在肾脏12。这个稿子我的目标s到提供有效的指导和见解的内源性物质的检测，如ATP的肾脏。 酶生物传感器微电极有超过测量分析物在体内的存在意味着许多优点。然而，特别的预防措施，应使用这种方法对于任何其他新方法。验证有一个既定的方法提供信心所获得的数据。例如，如先前所描述的26，27微透析测定制成。由传感器和那些从透析样品12得到确定的峰浓度之间没有观察到显著差异。微透析的局限性在于它仅测量分析物的稳态水平。这样，在细胞信号动态变化的评估只能通过使用酶的微电极的生物传感器的实现。的传感器响应时间的制造厂规格传感器之间是不同的类型。 体外传感器具有5-10秒和在 30-35秒，从10信号上升到90％ 的体内的传感器的响应时间。校准迹线（图6和7）表明的分析物添加/移除的时间分辨率。用于传感器和微透析方法既，需要使用特殊的酶阻断由分析物产生的信号，以确保特异性。 所描述的协议有几个挑战。正如任何传感器插入组织中，组织损伤确实发生。这能活化的信号通路可能影响测量28。为了尽量减少细胞损伤，更薄传感器将需要。然而，这是难以穿透肾囊与传感器所以针用于使一个小进入孔。薄的传感器更可能弯曲和破坏在插入其涂层。直径较大的传感器是更耐弯曲和resultant的损害他们的涂层。 图3表示薄和厚的传感器。除了厚传感器的耐弯曲，它们含有涂层的较厚的层，为酶层提供增加的保护。由于使用厚传感器组织损伤的关注是在肾脏大大低于这将是脑组织研究。插入深度近似和肾脏测量完成后的最终位置应进行验证。使用传感器头长度作为插入引导的皮质和髓质层之间估计足以作为传感器尖端为0.5毫米，由肾皮质是2-3毫米厚。结垢传感器也发生在血液中的更大的速率，从而限制了在长的传感器利用在体内的协议的一个实验。 1至1个半小时的录制时间，导致只有很小的传感器漂移。在评估减少传感器的性能是低灵敏度的主要标准到在校准过程中的分析物。增加的电噪声和基线电流的不稳定性也可以指示传感器端被损坏。为ATP的精确测量，两个传感器（ATP和空）应具有对信号进行进一步减法类似噪声和稳定性的特点。否则，传感器中的一个应更换。对于低噪声和检测小浓度分析物的，使用新的传感器对每个动物被建议。 几个步骤是成功的实验结果的关键。传感器是很脆弱的，应当谨慎处理以避免损坏传感器尖端。另外，补液后，传感器不应该暴露于空气中超过20秒。低噪音的录音都需要在组织中的成功侦破的生物信号。为了这个目的，一个高性能的实验室空气表和适当的电接地是必需的。加分析DUR的确切数量荷兰国际集团的校准步骤是必要的实验中准确的浓度测定。实现在离体肾手术肾好清算将导致成功的记录。酶的使用apyrase和过氧化氢酶在校准和在实验允许非特异性传感器灵敏度的评估和确认测量。 这些协议提供大大增强细胞外信号的细节肾脏。改进的灵敏度和时间分辨率由传感器提供可以允许我们解决变化的ATP在疾病状态信令和以下的药理操纵这在以前是无法检测。 在体内的协议是非常适合于复杂的生理研究，而离体是最适合于对肾脏药理应用的研究。这里使用的体内传感器的设计使得对在BL干扰前所未有电阻OOD灌注组织。两者合计，这些传感器提供了大量的，以前不可能与现有的协议和技术的应用程序。

酶促微电极的生物传感器（也引用为在本手稿传感器）已经用于在活细胞和组织学动态信令进程的宝贵工具。传感器提供在生物相关的浓度增加细胞信号分子的时间和空间分辨率。代替取样和分析在时间间隔过长时间采取细胞外液的，这些传感器响应尽可能快的酶反应而被分析物，由此产生实时测量1,2。间质性浓度的自分泌和旁分泌因子，如嘌呤或过氧化氢 ​​，和它们的释放动力学的快速检测可被用来建立一个轮廓为药物在正常和病理条件3的影响。目前，多数使用传感器的应用已经在脑组织切片和细胞培养物4-10。详见本稿件为目标的协议建立装置精确地测量在整个肾脏分析物的实时浓度。 以下协议被开发，研究间质性ATP和H 2 O 2信号在肾脏。在肾脏的天然环境中，外ATP迅速被内源性ectonucleotidases成它的衍生物（ADP，AMP和腺苷）分解代谢。此处所使用的传感器是高度选择性的ATP比其它嘌呤或ATP的降解产物11。这提供了一个很大的优势，因为它允许精确监测ATP释放和其信号功能的不断和动态的浓度。间质性ATP浓度是利用两个微电极一个Null传感器的组合，一个ATP传感器和测量。在组合的空传感器与过氧化氢 ​​酶的应用程序能够检测间隙H 2 O 2的浓度12。以下协议使用两种不同的浅草寺的设计具有的特性为最佳或者离体或在体内应用 RS。 这两种设计均基于甘油激酶和甘油-3-磷酸氧化酶的包含在传感器的酶层顺序催化反应，并通过ATP的存在驱动。在该组中的离体研究中所使用的传感器，H 2 O 2，最后的酶反应产物，通过氧化在铂/铱（铂-铱）线电极进行检测。传感器，用于在体内研究 ，而不是基于H 2 O 2的减少上介体包被的金电极设计用于血液灌注组织。 图 1所示是在这个手稿中描述这两种协议的方案。该空传感器是相同的其相应的ATP传感器除了它缺乏结合酶。因此，除了与H 2 O 2检测的过氧化氢 ​​酶，所述空传感器MEA祖雷斯贝尔非特异性干扰。 ATP的浓度通过减去空检测非特异性干扰和背景的 H 2 O 2的从ATP的传感器信号来计算。几个传感器也是可商购时与相应的空成对检测其它被分析物，包括腺苷，ionosine，次黄嘌呤，乙酰胆碱，胆碱，谷氨酸盐，葡萄糖，乳酸盐，D-丝氨酸用于离体应用或腺苷，ionosine，和次黄嘌呤用于体内传感器。 传感器的精确检测分析物的能力取决于适当的前后的校准13。这确保了分析占生物组织中使用期间发生的漂移的传感器灵敏度。传感器保持甘油被用作在传感器的酶反应的试剂贮库。如果传感器未在含有甘油浴溶液中使用，将洗出随着时间的推移。短řECORDING时间然后要尽量减少传感器漂移。此外传感器内源性蛋白酶和蛋白片段结垢可以大大降低传感器 14的灵敏度。 本手稿确立使用酶促微电极的生物传感器用于离体和体内肾脏制剂。实时定量分析提供了前所未有的，可能揭示新的见解肾脏疾病和药物制剂的机制细胞信号细节。

<table><tbody><tr><td>Sensor Kit</td><td>Sarissa Biomedical</td><td>SBK-ATP-05-125</td><td>The kit includes storage bottle, rehydration chamber, electrode leads, and reference electrodes.&amp;nbsp; Also included with the kit is the user&#039;s choice of sensors.</td></tr><tr><td>Sarissaprobe ATP Biosensor 125 &amp;mu;m</td><td>Sarissa Biomedical</td><td>SBS-ATP-05-125</td><td>store at 2-8 &lt;sup&gt;o&lt;/sup&gt;C before use</td></tr><tr><td>Sarissagold ATP Biosensor 50 &amp;mu;m</td><td>Sarissa Biomedical</td><td>SGS-ATP-10-50</td><td>store at 2-8 &lt;sup&gt;o&lt;/sup&gt;C before use</td></tr><tr><td>Sarissaprobe null sensor 125 &amp;mu;m</td><td>Sarissa Biomedical</td><td>SBS-NUL-20-125</td><td>store at 2-8 &lt;sup&gt;o&lt;/sup&gt;C before use</td></tr><tr><td>Sarissagold null sensor 50 &amp;mu;m</td><td>Sarissa Biomedical</td><td>SGS-NUL-10-50</td><td>store at 2-8 &lt;sup&gt;o&lt;/sup&gt;C before use</td></tr><tr><td>Sarissaprobe ATP Manual</td><td>Sarissa Biomedical</td><td></td><td>&lt;a href=&quot;http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf&quot;&gt;http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf&lt;/a&gt;</td></tr><tr><td>Faraday cage&amp;nbsp;</td><td>TMC</td><td></td><td></td></tr><tr><td>Dual channel potentiostat</td><td>Digi-Ivy</td><td>DY2021</td><td>Type II Faraday cage</td></tr><tr><td>Data acquisition program</td><td>Digi-Ivy</td><td>DY2000</td><td></td></tr><tr><td>Perfusion pump</td><td>Razel Scientific Instruments</td><td>Model R99E</td><td></td></tr><tr><td>Fiber optic illuminator</td><td>Schott</td><td>ACE 1</td><td></td></tr><tr><td>micromanipulator</td><td>Narishige</td><td>MM-3</td><td></td></tr><tr><td>micromanipulator magnetic stand</td><td>Narishige</td><td>GJ-8</td><td></td></tr><tr><td>air table</td><td>TMC</td><td>63-500</td><td></td></tr><tr><td>isoflurane ventilator</td><td>LEI Medical</td><td>M2000</td><td></td></tr><tr><td>3 ml petri dish</td><td>Fisher Scientific</td><td>S3358OA</td><td></td></tr><tr><td>needle</td><td>Santa Cruz</td><td></td><td>26-30 G</td></tr><tr><td>pins</td><td></td><td></td><td>Standard dissection pins</td></tr><tr><td>catheter</td><td></td><td></td><td>Polyethylene tubing (PE50)</td></tr><tr><td>catheter tissue glue</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>suture</td><td>Look</td><td>SP117</td><td></td></tr><tr><td>rubber bands</td><td></td><td></td><td>any 2-4 mm wide rubber bands</td></tr><tr><td>silicone</td><td>Momentive</td><td>RTV-615 Clear 1#</td><td></td></tr><tr><td>clamp</td><td>Fine Science Tools</td><td>18052-03</td><td></td></tr><tr><td>standard dissection kit</td><td></td><td></td><td>Kit should include scalpel and dissection sissors&amp;nbsp;</td></tr><tr><td>Kidney Cup</td><td></td><td></td><td>Of own design</td></tr><tr><td>standard chemicals</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A6559-25UMO</td><td>100 mM ATP solution</td></tr><tr><td>hydrogen peroxide</td><td>Sigma-Aldrich</td><td>216763</td><td></td></tr><tr><td>glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Apyrase</td><td>Sigma-Aldrich</td><td>A7646</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>isoflurane</td><td>Clipper</td><td>10250</td><td></td></tr><tr><td>inactin</td><td>Sigma-Aldrich</td><td>T133</td><td></td></tr><tr><td>ketamine</td><td>Clipper</td><td>2010012</td><td></td></tr><tr><td>Hanks Balanced Salt Solution&amp;nbsp;</td><td>Gibco</td><td>14025092</td><td></td></tr></tbody></table>

use of enzymatic biosensors to quantify endogenous atp or h2o2 in the kidney

酶促微电极的生物传感器已被广泛地用于测量实时外信号。大部分它们的使用一直局限于脑切片和神经元细胞培养物。最近，这项技术已被应用到的整个器官。在传感器的设计的发展使得可能细胞信号传导在血液灌注的体内肾脏的测量。本协议列出来衡量ATP和H 2 O 2信号在大鼠肾间质所需的步骤。两个独立的传感器设计用于离体和体内的协议。这两种类型的传感器都涂有上的选择渗透性层的顶部上的薄酶促生物层，得到快速响应，灵敏的和选择性的生物传感器。选择渗透性层保护从生物组织中的干扰物的信号，并且所述酶层利用甘油激酶和甘油-3-磷酸的连续催化反应在ATP的存在phate氧化酶产生的 H 2 O 2。设定用于体外研究的传感器的进一步检测的分析物的 H 2 O 2的氧化上的铂/铱（铂-铱）线电极。传感器用于在体内研究 ，而不是基于对H 2 O的2上的介体包被的金电极设计用于血液灌注组织的减少。最终浓度变化是由实时电流分析法随后校准到已知浓度的分析物的检测。此外，安培信号的特异性可通过加入酶如过氧化氢 ​​酶和apyrase该分解的 H 2 O 2和ATP相应的确认。这些传感器还很大程度上依赖于在每次试验后，精确的校准。下面的两个协议建立实时检测的ATP和H 2中的研究O 2在肾组织，并且可以进一步修饰，以延长所述方法为在其他生物制剂或整个器官的使用。

酶促微电极的生物传感器的设计允许实时检测在整个肾脏分析物。一般实验设计为或者离体或体内研究中示出使用图1 .The传感器和外科手术会因在该研究是离体或在体内 。 以获得可重复性的结果，精确前后的校准是重要的。 图6A示出在校准从体外 ATP的传感器看到的信号的代表跟踪。需要注意的是apyrase迅速消除了ATP信号。过氧化氢 ​​酶对ATP的信号没有影响，表明了其特异性的H 2 O的2它创建（图6B）。校准过程产生被用来计算ATP的动态变化（图6A，右图）的线性拟合。 在viVO传感器校准产生了类似跟踪到体外的传感器。然而，该传感器检测的还原，而不是氧化电流，因此产生的电流是负的。这些传感器的校准也产生在0.3至80微米的范围内的线性拟合（图7A 右图）。 体内传感器对ATP比其它嘌呤产品的特异性示于图7B。 这里所描述的方法使我们能够衡量对药物输注12的内源性物质和急性两种变化的基础水平。 示于图11是血管紧张素II诱导的H 2 O 2的内源性间质性浓度的盐抗性和盐敏感大鼠的变化。对于这些实验，新鲜分离肾脏从只SD（SD）或Dahl盐敏感（SS）的大鼠下恒定层流（650微升/分钟）灌注经1μM的血管紧张素Ⅱ。由于笑WN 在图11中，血管紧张素Ⅱ诱导H 2 O 2从SD和SS大鼠肾脏急性释放。然而，每个响应的最大幅值中的SS大鼠中显著升高，特别是当动物喂以高盐饮食12。 如图 12所示是在体内的生物传感器的一个典型应用。过氧化氢 ​​酶（5微克/毫升）由生物传感器检测到的完全阻断了的 H 2 O 2的信号的输注。这些实验说明使用特定酶的生物传感器结合安培技术的内源性物质和它们的释放的实时测量基础水平的响应于药物干预检测的。这种方法的进一步应用研究嘌呤信号和过氧化氢的作用将提高我们的肾脏病如盐敏感性高血压的认识和了解，肾牛idative应力和慢性肾脏疾病。 <img alt="图1" src="/files/ftp_upload/53059/53059fig1.jpg" /> 图1原理图议定书的校准与感兴趣的分析物和测试其特异性完成之前立即实验的开始。 体内协议应遵循复杂的生理实验和体外药理学的应用。实验后校准应做数据分析时予以考虑。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig1large.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/53059/53059fig2.jpg" /> 图2。酶微电极传感器，两个用于离体应用传感器尺寸被示出，50微米和125微米的直径。每个传感器导线延伸进入毛细管连接到金端连接器（未示出）。插图显示了传感器顶端涂有酶ATP检测。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig2large.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/53059/53059fig3.jpg" /> 图3。双通道恒电位仪，标有CH1和CH2的恒电位通道。 1）CH1 ATP的传感器连接和2）CH2的空传感器连接3）连接的电耦合到参比电极。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 屁股="jove_content"FO:保持together.within页="总是"&gt; <img alt="图4" src="/files/ftp_upload/53059/53059fig4.jpg" /> 图4。传感器安装程序。在一个法拉第笼，以减少电噪声和对无振动的工作表面的高性能实验室表进行数据采集。 1）一个温度控制的外科表用于保持在生理实验2动物体温）的微操作被实验3）的光源是需要外科手术和传感器插入4期间附连到磁性安装适配器传感器的灵活定位）双通道电位5）持有的传感器。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> p_upload / 53059 / 53059fig5.jpg"/&gt; 图5。循环伏安法对于 体外研究，传感器循环伏安法为-0.5和0.5 V之前，校准协议之间进行10次。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53059/53059fig6.jpg" /> 图6。电流分析法校准用于离体。（A）的校准使用加法已知的ATP浓度向浴溶液中。通讯记录在渐近线水平（黑色线）安培值。所获得的电流产生一个校正公式。一个例子显示在右侧面板中。红色曲线是真正水流核苷酸的空传感器响应仅加入的 H 2 O 2的。 （B）中的空传感器校准通过加入已知的 H 2 O 2的浓度（红色箭头）向浴溶液和安培值记录渐近线（细黑箭头）。除了过氧化氢酶的槽液（粗黑箭头）导致快速电流衰减。右侧面板显示空传感器校准方程。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53059/53059fig7.jpg" /> 图7。电流分析法校准;以类似的方式进行的体内校准的体内电极到该详细的体外研究除还原反应引起的反向电流（极性）。 （A）中加入已知的ATP浓度的产生的ATP传感器（黑色迹线）安培电流，但对空传感器（红色迹线）没有影响。此外apyrase的熄灭ATP的传感器的电流。 （B）的ATP的传感器的特异性通过加入10μM的不同嘌呤剂（双绞线，UDP和腺苷）的确认。 ATP的进一步的应用提供了稳定的可检测的安培的电流。根据调解员涂金电极体内的传感器（C）的显微照片。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig7large.jpg" target="_blank">请点击此处查看本figu的放大版本。</a>回覆。 <img alt="图8" src="/files/ftp_upload/53059/53059fig8.jpg" /> 图8。肾杯，在 体内研究 ，肾脏被保持仍在使用中所示的不锈钢肾脏杯。两种尺寸杯子用于容纳肾脏大小的变化。这些杯子减少这是由动物的呼吸所产生的运动伪影。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/53059/53059fig9.jpg" /> 图9. 离体分离并灌注肾脏。将分离的肾放入一个1）培养皿涂有厚厚的SIlicone底部销插入2）实验3）ATP的传感器在肾动脉进行插管并连接于注射器泵用于恒定灌注，4）和Null传感器被插入到参考电极放置在靠近浸没肾脏肾5）进入槽液。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/53059/53059fig10.jpg" /> 图10. 体内血液灌流肾脏。（A）的左肾暴露，并放置在肾杯，右肾保持完整的动物内。两个传感器被插入到肾。 BSA:氯化钠经由插管颈静脉输注以抵消流体损失造成by的插入体内实验的大中线切口（B）的实施例与植入间质性导管用于直接的药理学应用1）肾脏是由肾脏杯2）ATP传感器3）空传感器和4）参比电极保持入肾5）导管植入的肾脏和连接到蠕动泵的层流药物输注的腹面。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。请点击此处查看该图的放大版本。</a> <img alt="图11" src="/files/ftp_upload/53059/53059fig11.jpg" /> 的H 2图11. 离体分析 </<html>子&gt; 肾脏O 2。血管紧张素Ⅱ灌注导致H 2 O 2的释放在大鼠肾皮质。 （ 一）平均的实时变化H 2 O 2浓度（灰条显示标准误差）总数为N = 8的应用程序（从4个不同的4只肾）。顶部条表示血管紧张素Ⅱ的应用。 （B）的最大H态期间，血管紧张素Ⅱ灌注只SD（标清）和Dahl盐敏感（SS）大鼠2 O 2浓度振幅值喂食低，高盐饮食，分别。 * - P &lt;0.05与SD大鼠。这个数字是改编自参考12的许可。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /files/ftp_upload/53059/53059fig12.jpg"/&gt; 图12. 在 H 2 O的2。 在体内评估间质性ħ的实施例的体内分析 2 O 2的浓度在大鼠的SS的髓质馈送低盐饮食（ 见图10B）。过氧化氢 ​​酶通过植入导管为5分钟间隔的间隙应用产生于肾脏髓质的 H 2 O 2的信号的一个完整的封锁。减少过氧化氢 ​​酶，从冲洗肾血流量，导致了信号，这是由一个额外的过氧化氢 ​​酶的应用（10分）再次受阻的部分恢复。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53059/53059fig12large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney at JoVE.com

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

利用酶生物传感器的量化内源性ATP或H<sub&gt; 2</sub&gt; 0<sub&gt; 2</sub&gt;在肾脏

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

use-enzymatic-biosensors-to-quantify-endogenous-atp-or-h2o2

Universidad San Sebastian

Research

JoVE Journal

Biology

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

11.9K 次观看.  Medical College of Wisconsin. Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney. 

视频: Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

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

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

生物化学

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 ...

生物工程

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

生物学

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

测量<em&gt;体外</em&gt;的酶学性质ATP酶活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-&#946;), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging.
This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-&#946;, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-&#946; in&#160;a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-&#946;-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes ...

使用 mtHyper7 对活酵母细胞中的线粒体过氧化物进行成像

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

作者聚焦:推进线粒体研究 - 用于亚细胞分析的 mtHyper7 生物传感器 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

利用酶生物传感器的量化内源性ATP或H

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

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

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

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

纳米传感器检测体内蛋白酶活性的无创诊断

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

一个简单的生物测定血管内皮生长因子的评价

测量

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

利用酶生物传感器的量化内源性ATP或H

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

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

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

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

纳米传感器检测体内蛋白酶活性的无创诊断

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

一个简单的生物测定血管内皮生长因子的评价

测量

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的