这项研究是由基础科学研究计划通过教育，科学与技术部（2013068067）资助的韩国国家研究基金会（NRF）的支持;由教育部，科技部，首尔国立大学医院，高血压的韩国社会（2013年），SK电信研究基金的韩国部脑韩国21毕业生计划（编号3420130290）和中国国家自然科学基金（NSFC 31460265;国家自然科学基金81260035）。

该协议是按照指南卫生的联合国国家机构发布的实验室动物的护理和使用（NIH出版号85-23，1996年修订）。它是经首尔国立大学的机构动物护理和使用委员会（IACUC）（IACUC批准号:SNU-101213-1）。 1.缓冲液配制（表材料和设备） <ol><li>制备300毫升新鲜分离溶液对实验当天（以mM:氯化钠，135;氯化钾，5.4; MgCl 2的，3.5;葡萄糖，5; HEPES，5; 的 Na 2 HPO 4，0.4;牛磺酸，20; pH值7.4用NaOH）。 </li><li>制备100毫升新鲜的存储溶液在实验当天（以mM:NaCl的120;氯化钾5.4; MgSO 4上5; 氯化钙 0.2;钠丙酮酸5;葡萄糖5.5;牛磺酸20; HEPES 10;甘露醇29; pH 7.4的用NaOH）。 </li><li>制备灌注溶液1L（以mM:氯化钠，141.4;氯化钾，4;和NaH 2 PO 4，0.33; MGCL 2，1; HEPES，10;葡萄糖，5.5; 氯化钙 ，1.8;甘露醇，14.5; pH为7.4，用NaOH）。 </li><li>通过加入胶原酶（1毫克/毫升），蛋白酶（0.1毫克/毫升），牛血清白蛋白（BSA，1.67mg / ml）和钙离子 （0.05毫摩尔）制备加入30ml胶原酶溶液1至25隔离毫升解。 </li><li>通过加入胶原酶（1毫克/毫升），牛血清白蛋白（1.67毫克/毫升），和钙离子 （0.05毫摩尔）至16.7毫升隔离溶液制备20毫升胶原酶溶液2。 </li><li>通过加入0.4克的BSA至40ml的隔离溶液制备的40ml BSA溶液。单独10毫升和30ml成两个烧杯。添加的 Ca 2+至30毫升BSA溶液，使最终的Ca 2+浓度的BSA溶液是1毫米。 </li></ol> 2.准备左心室（LV）心肌细胞的隔离<ol><li>从动物设施的准备和隔离室转让清洁运输笼8-12周龄，雄性SD（SD）大鼠。 </li><li> H吃两水浴至37℃。 注意:使用一种水浴水 - 夹套容器和的Langendorff灌注系统的灌注管。使用其他水浴以分离单个细胞搅动心肌组织的树干。 </li><li>加入5 ml和3.3的BSA溶液（不含Ca 2+）至25毫升胶原酶溶液1和16.7 ml胶原酶溶液2 ml至补足体积至30ml和20ml，分别。 </li><li>在的Langendorff灌注系统，分别为（ 图1A）添加隔离溶液（100毫升）和胶原酶溶液1（30毫升）中，以第1列（第1栏）和第2列（第2列）。 </li><li>充氧在的Langendorff灌注系统（ 图1A） 通过氧连接管分离溶液和胶原酶溶液1中第1栏和Col.2。同样地，含氧化合物胶原酶溶液2，并在摇动的水槽用100％O 2的经由所述BSA溶液氧气连接管。 </li></ol> 3. LV心肌细胞的分离<ol><li>麻醉通过戊巴比妥钠（30毫克/千克）的腹膜内注射大鼠和脚趾捏和缺乏撤回反射的确认麻醉状态。 </li><li>移动老鼠解剖盘。在仰卧位，固定四条腿到主体用胶带的侧面。 </li><li>应用胸中期轴向切口手术剪刀打开胸腔，确保不损害心脏。使用另一一双干净的剪刀从血管连接（ 例如，上，下腔静脉，肺血管，主动脉）和心包膜13解剖心脏。 </li><li>留足够长的主动脉的一部分（5 - 8 mm产品），夹紧用细镊子主动脉，和1分钟（ 图1A）内快速安装的Langendorff灌注系统的插管。领带缝合线4/0紧紧过主动脉。 </li><l我&gt;阀门打开上校1和10分钟预热和含氧隔离解决方案（灌注率:12-14毫升/分钟）灌注离体心脏。关闭阀上的第1栏，打开阀门上西2和8胶原酶溶液灌注1的心脏 - 10分钟。 </li><li>通过削减主动脉卸除消化心脏，并通过召开用钳子将主动脉含有新鲜的隔离解决方案（ 图1Bi）烧瓶中传送。用细剪刀和镊子大部分LV游离壁（包括隔膜），以切成小块（〜22毫米， 图1Bii）。 </li><li>转移片放入含预热和含氧的新鲜胶原酶溶液2（ 图1Biii）的烧瓶中。振摇10分钟。请提供氧气的含肌细胞胶原酶溶液2。 </li><li>移动心肌细胞-悬浮液10 ml离心管中，使用滴管带吸球，并添加钙 -含BSA溶液（1:1的体积比）。离心在30克2分钟，弃上清。重悬在5ml BSA溶液的肌细胞沉淀。离心并弃去上清液。 </li><li>驱散心肌细胞颗粒，并在RT（ 图1Biv-VI）保持心肌细胞在10毫升预氧化的存储解决方案。 </li><li>在烧瓶内的剩余的LV组织 - 重复程序（3.9步骤3.7）。 注意:不断重复的过程（步骤3.7 - 3.9）再次直到大部分LV组织消失，细胞获得了良好的收益。 </li><li>保持在存储溶液中的细胞在室温6-8小时，直至实验结束。 </li></ol> 4.细胞内钙瞬变和心肌细胞收缩的同时测量<ol><li>负载的LV心肌细胞与乙酰氧甲基酯的Fura-2 AM（2μM）。 注:在黑暗的管（ 图1Bvii）装载的细胞执行所有加载的程序和实验。 <ol><li> CENTRifuge在2000 xg离心肌细胞悬浮液（1ml）中，持续10秒。弃去上清液并重新悬浮于1ml台罗德氏溶液具有低Ca 2+浓度（250μM，表材料和设备）的肌细胞沉淀。 </li><li>添加的Fura-2AM和泊洛沙姆407（2微升），轻轻分散肌细胞悬浮液，并保持该混合物在RT（20 - 24℃）15分钟（ 图1Bvii）。 </li><li>离心混合物，持续5秒，弃去上清液并分散肌细胞沉淀在1ml含有500μM 的 Ca 2+灌注溶液。 10分钟后，离心该混合物，持续5秒，并丢弃上清液。 </li><li>加入新鲜的灌注溶液（500μM 钙 1毫升），并保持的Fura-2AM -加载 ​​心肌细胞颗粒在此解决方案的记录。 </li></ol></li><li> LV的细胞收缩和细胞内Ca 2+测定<ol><li>录像之前，填补通过运行输液管与台氏溶液，这是水套预热到36℃。 </li><li>放置的Fura凌晨2点几滴 - 加载的LV心肌悬浮于5倒置荧光显微镜的腔室 - 8分钟。慢慢灌注台罗德氏溶液（2.2毫升/分钟）。 </li><li>按数字刺激开始字段刺激（2赫兹）的前面板上的"启动"按钮。 注意:输出电压（10V，5毫秒的持续时间）通过设置在腔室的任一侧铂导线施加到心肌细胞中的腔室。 <ol><li>选择细胞的合同稳定（而不是那些显示高血糖或低收缩）记录。 </li></ol></li><li>调整选择的心肌细胞中的基于视频的肌节检测系统的水平位置和调整显微镜的焦点，以获得肌节的最佳图像。在其中肌节清晰明确，直到平均观察到的区域紫色矩形框的位置肌节长度（红色峰）的显示单一尖峰（ 图2A，下图）。 </li><li>记录响应于场刺激（ 图2A和B）小节的长度的变化。 注意事项:1范围内使用加载的细胞 - 2小时。 </li><li>调节摄像机的光圈，使得在基于视频的肌节的检测系统中的场是心肌细胞（ 图2A）的尺寸。刺激与360纳米/ 380纳米激发和发射510纳米（收购频率1000赫兹）的心肌细胞。记录肌节缩短和与场刺激（ 图2B）的Fura2调幅比。 </li></ol></li></ol> 5.肌丝钙离子敏感性评价<ol><li>平均肌节长度和Ca在稳态2+瞬变（10 - 20痕量）和绘制的Fura-2比对相同的心肌细胞的肌节长度的相平面环（都与测量值S和增量变化; 图2C）。 </li><li>在每个小区，定义的Fura -2在50％松弛（EC 50， 图2C）的比例。比较这两个循环，每个干预的EC 50。 </li></ol>

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The authors declare that they have no competing financial interests.

这里，我们描述了协议，以评估肌丝的 Ca 2+在单个孤立心肌细胞的敏感性的变化，并强调测定该参数一起电生理特性，细胞内Ca 2+瞬变，和肌丝动力学的重要性。这是因为一个或两个参数的记录可能没有阐明心脏收缩和舒张的基本机制。不同于测量肌细胞收缩和细胞内Ca 2+信息单独1的常规方法，本方法检查这两个参数在相同的心肌细胞同时。 <...

心肌兴奋收缩（EC）耦合是分析心肌， 即心脏1,2的收缩功能的机械性能的根本方案。 EC耦合 ​​通过膜去极化继发于细胞膜离子通道的活动发起（ 例如，电压门控钠通道，它可以通过膜片钳技术测量）。电压门控的L型的随后活化的Ca 2+通道（LTCCS）和Ca 经由 LTCCS 2+内流触发散装的Ca 2+通过兰尼碱受体（RyRs的）的释放，从纳摩尔提高胞质Ca 2+浓度（纳米）到微摩尔（μM）的水平。这种增加在细胞内钙离子促进钙离子结合在薄丝肌钙蛋白C（TNC）和抒发灯丝复杂的构象变化，促进肌动球蛋白相互作用和达到myocardi人收缩3。相反，细胞内钙重新摄取回SR中的肌质网（SR）通过的 Ca 2+ -ATP酶（SERCA2a的），或者是通过 钠 / 钙交换和质膜挤出肌纤维钙离子 ATP酶1,2。因此，在细胞内的Ca的下降2+指使细长丝的构象变化返回到原来的状态，从而导致肌动蛋白-肌球蛋白和肌细胞松弛1-3的解离。在这个方案中，SERCA2a的活性通常被认为是，以确定心肌松弛的速度，因为它占70 -在大多数哺乳动物的心脏细胞1细胞内的Ca 2+去除的90％。正如LTCC，碱受体和SERCA2a的等这样的，不正常的钙处理一直被认为是受损的收缩和放松患病心脏1-4的主要机制。 离子 ，其功能如同在EC耦合 ​​账户信使约1％的总细胞内Ca 2+和广大钙势必细胞内钙离子缓冲5,6。这是由于这样的事实，即各个的 Ca 2+缓冲液是在心肌细胞丰富， 例如，膜磷脂，ATP，磷酸，钙调蛋白，小清蛋白，肌原纤维TNC，肌球蛋白，SERCA2a的，并且在对SR肌集钙蛋白。5,6,7。其中，SERCA2a的和跨国公司是主要的钙缓冲5,6,7。此外，钙离子结合到其缓冲器是抽搐期间一个动态的过程（ 例如，钙2+ 30-50％结合于肌钙蛋白和Ca期间将它解离2+瞬变7）和中的 Ca 2+结合的原因的其他变化在细胞内钙的改变游离钙离子的"释放"到细胞质，结果2+浓度entration。因此，细胞内Ca 2+水平的扰动引起异常肌丝的运动，这是收缩功能障碍和心律失常8,9的前体。许多因素（包括生理和病理）可以是肌丝蛋白质的转录后修饰，影响肌丝的 Ca 2+缓冲和肌丝的 Ca 2+敏感性8-10的来源。最近，据报道，在肌丝蛋白突变增加的 Ca 2+结合亲和力和细胞内Ca 2+的处理，触发的 Ca 2+瞬变，异常的 Ca 2+释放，和心律失常8的暂停依赖性增强。根据这一理念，我们也表现出高血压大鼠心脏继发于神经元型一氧化氮合酶上调的肌丝钙脱敏与高架舒张和收缩的 Ca 2+水平有关11，这又增加了LTCC的对于Ca 2+依赖性失活12的脆弱性。因此，肌丝的 Ca 2+敏感性是细胞内Ca 2+稳态和肌细胞收缩功能的"有效的"调节器。它已成为必要分析肌丝和Ca之间的相互作用2+处理的蛋白质为心肌兴奋收缩耦联及心功能的彻底调查。 在这里，我们描述了评估肌丝钙离子敏感性在孤立的心肌细胞变化的协议。细胞内钙离子分布的综合分析，肌丝钙离子敏感性和收缩会发掘新机制基本心肌力学。

<table border="0" cellpadding="0" cellspacing="0" width="1136">
	<tbody>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">Sprague Dawley rat</td>
			<td style="width:131px;">Koatech</td>
			<td style="width:125px;"></td>
			<td style="width:565px;">8-12 weeks</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:315px;">Pentobarbital Sodium</td>
			<td style="width:131px;">Hanlim Pharmaceutical (Korea)</td>
			<td style="width:125px;">AHN901</td>
			<td style="width:565px;">Insurance code:645301220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Taurine</td>
			<td style="width:131px;">Merck</td>
			<td style="width:125px;">8.08616.1000</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fura-2 (AM)</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:125px;">F1221</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pluronic F127 20% solution in DMSO</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:125px;">P3000MP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking Water Bath</td>
			<td style="width:131px;">Chang Shin Scientific</td>
			<td style="width:125px;">Model: C-108</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:315px;">IonWizard Softwae Suite</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;">Experimental Builder</td>
			<td style="width:565px;">Acquisition and Analysis of EC Coupling Data in Myocytes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:315px;">Myocyte Calcium &#38; Contractility Recording System</td>
			<td style="width:131px;">IonOptix Ltd</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Circulating Water Bath</td>
			<td style="width:131px;">BS-Tech</td>
			<td style="width:125px;">BW2-8</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:315px;">Myocyte Fluorescence Microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:125px;">DIATPHOTO 200</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:315px;">MyoCam-S Power</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="36">
			<td height="119" rowspan="3" style="height:119px;width:315px;">Fluorescence &#38; Video Detection</td>
			<td rowspan="3" style="width:131px;">IonOptix</td>
			<td style="width:125px;">MyoCam-S</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:125px;">CFA300</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:125px;">PMT400</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Fluorescence &#38; System Interface</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">FSI700</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Excitation Light Source</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">mSTEP</td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">High intensity ARC Lamp Power supply</td>
			<td style="width:131px;">Cairn Reseach</td>
			<td></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:315px;">Filter wheel controller</td>
			<td style="width:131px;">IonOptix</td>
			<td style="width:125px;">GB/MUS200</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;">Digital Stimulator</td>
			<td style="width:131px;">Medical Systems Corportion</td>
			<td>S-98 Mutimode</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compositions of Experimental Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name</td>
			<td style="width:131px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:565px;">Comments</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:315px;">Isolation Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 135</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 3.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Na2HPO4&#160;</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">71649</td>
			<td style="width:565px;">Concentration (mmol) 0.4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Storage Solution (pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 120</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 5.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.2</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 29</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgSO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M5921</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Sodium Pyruvate</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P2256</td>
			<td style="width:565px;">Concentration (mmol) 5</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Taurine</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">CB2742654</td>
			<td style="width:565px;">Concentration (mmol) 20</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:315px;">Perfusion Solution (Tyrode solution, pH: 7.4, NaOH)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S9625</td>
			<td style="width:565px;">Concentration (mmol) 141.4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">KCl</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P4504</td>
			<td style="width:565px;">Concentration (mmol) 4</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">NaH2PO4</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">S8282</td>
			<td style="width:565px;">Concentration (mmol) 0.33</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">HEPES</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">H3375</td>
			<td style="width:565px;">Concentration (mmol) 10</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Glucose</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">G8270</td>
			<td style="width:565px;">Concentration (mmol) 5.5</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td>Concentration (mmol) 1.8&#160;&#160;&#160;&#160;&#160; For Fura 2AM loading, CaCl2 concentrations are 0.25 mM and 0.5 mM</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">MgCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">M2001</td>
			<td style="width:565px;">Concentration (mmol) 1</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Mannitol</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">M4125</td>
			<td style="width:565px;">Concentration (mmol) 14.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 1</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (30mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 5 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Protease</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">P6911</td>
			<td style="width:565px;">Concentration (mmol) 0.1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Collangenase Solution 2</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (20mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin (BSA solution 3.3 mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;">Concentration (mmol) 1.67 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Collagenase Type 2</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:125px;">LS004177</td>
			<td style="width:565px;">Concentration (mmol) 1 mg/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 0.05 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">BSA solution</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Isolation Solution (40mL)</td>
			<td style="width:131px;"></td>
			<td style="width:125px;"></td>
			<td style="width:565px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">Bovine Fetal Albumin</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:125px;">A7906</td>
			<td style="width:565px;">Concentration (mmol) 400 mg</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:315px;">CaCl2</td>
			<td style="width:131px;">Biosesang</td>
			<td style="width:125px;">C2002</td>
			<td style="width:565px;">Concentration (mmol) 1mM</td>
		</tr>
	</tbody>
</table>

assessment of myofilament ca2+ sensitivity underlying cardiac excitation-contraction coupling

心脏衰竭和心律失常是全世界发病率和死亡率的主要原因。然而，在患病心脏发病和心肌故障的机制仍有待完全澄清。最近令人信服的证据表明，在肌丝的 Ca 2+敏感性的变化会影响细胞内Ca 2+稳态和离子通道活动在心肌细胞，负责在健康和患病心脏心脏动作电位和收缩的主要机制。事实上，潜在的心脏动作电位（如钠离子 ，钙离子和钾离子通道，钠+ -Ca 2+交换）和细胞内钙离子处理的蛋白质（ 例如 ，兰尼碱受体和钙离子通道的活动和运输2+ -ATP酶肌浆网（SERCA2a的）或磷蛋白和磷酸化其）的常规测量evalu吃了心脏兴奋收缩（EC）耦合的基本机制。在细胞膜和细胞内钙离子的变化包括电活动有兴奋收缩耦联的触发信号，而肌丝的收缩和舒张的功能单位，肌丝钙离子敏感性是在肌原纤维性能的实施势在必行。然而，很少有研究纳入肌丝钙离子敏感性到心肌的功能分析，除非它是研究的重点。在这里，我们描述了测量肌缩短/再延长使用的Fura-2 AM（比率检测），并评估肌丝的 Ca 2+从大鼠心脏心肌细胞敏感性的变化，细胞内钙离子水平的协议。其主要目的是强调肌丝钙离子敏感性，应考虑到EC联轴器机械分析。我的全面排查对背后的健康和患病心脏肌细胞收缩将提供有价值的信息设计平移和治疗价值的更为有效的战略通道，离子转运，细胞内钙离子处理和肌丝钙离子敏感性。

LV肌细胞正常和高血压大鼠心脏分离。有明确的条纹（代表肌节），并稳定收缩响应于场刺激棒状细胞被认为是最佳的细胞和被选择用于记录（ 图2A）。在F中igure 2A所示的例子中，一个的Fura上午02点-loaded左心室心肌细胞水平定位并调节摄像机的光圈，使心肌细胞占据了大部分的记录区和最小的背景区域被包括在内。在录制现场，通过点击和拖动?...

Watch this Scientific Journal Video about Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling at JoVE.com

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

肌丝的Ca评估<sup&gt; 2+</sup&gt;灵敏度潜在心脏兴奋收缩耦联

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

assessment-myofilament-ca2-sensitivity-underlying-cardiac-excitation

Research

JoVE Journal

Biology

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

9.4K 次观看.  Seoul National University College of Medicine. This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts. 

视频: Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

调查的基本免疫机制，器官移植排斥反应

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...