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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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...

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 border="0" cellpadding="0" cellspacing="0">
	<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.&#160; Also included with the kit is the user&#39;s choice of sensors.</td>
		</tr>
		<tr>
			<td>Sarissaprobe ATP Biosensor 125 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SBS-ATP-05-125</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissagold ATP Biosensor 50 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SGS-ATP-10-50</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissaprobe null sensor 125&#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SBS-NUL-20-125</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissagold null sensor 50 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SGS-NUL-10-50</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissaprobe ATP Manual</td>
			<td>Sarissa Biomedical</td>
			<td></td>
			<td><a href="http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf">http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf</a></td>
		</tr>
		<tr>
			<td>Faraday cage&#160;</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 gauge</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&#160;</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&#160;</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。?...

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

Research

JoVE Journal

Biology

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

11.8K 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

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

SC1（Pluripotin）使用，以支持在没有LIF的自我更新MESC

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

科研

生物学

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...