此外，我们感谢在海德堡大学的心脏外科医生提供人类心房组织和克劳迪亚Liebetrau卡特琳Kupser和雷蒙娜格尔其过硬的技术支持。还要特别感谢安迪·老特拉福德的有益的建议和意见期间建立的Ca 2 +瞬时测量。作者希望表达他们最深切的谢意德累斯顿科技大学的药理学和毒理学系（头：乌苏拉乌鸦）的成员，他们为我们提供了学习的基本方法和技巧，在细胞电生理和心肌隔离的机会。 作者的研究支持德国研究基金会（Do769/1-1-3），德国联邦教育和研究部通过的心房颤动能力网络（01Gi0204）和德国心血管研究中心，欧盟通过在欧盟ropean转化研究心房颤动（EUTRAF网络，FP7-HEALTH-2010年，大型集成项目的议案“，第261057号）和美国欧洲北房颤研究联盟（ENAFRA）授予的基金会Leducq（07CVD03）。 在视频文件中所示的原理概述使用施维雅医疗艺术。

实验协议需要由当地伦理委员会批准，所有患者需要给予书面知情同意书。我们的研究是曼海姆医学院伦理委员会的海德堡大学（2011-216N-MA）的批准，并符合所有人类福利机构，国家和国际准则进行。所有患者的书面知情同意书。 0。获取人类心房组织在开放式心脏手术体外循环移植的患者在常规插管过程中，右心耳的尖端通常去除，可用于隔离心房心肌细胞。切除后的组织样本被立即转移到50毫升猎鹰管，用无菌的Ca 2 +免费运输解决方案，含有2,3 -丁二酮肟（ 见表一 ; BDM，收缩抑制剂，防止心肌Çontracture）。在30和45分钟之间的运输时间，我们并没有认识到孤立的心房心肌数量和质量方面具有明显的优势，冷却或充氧。在一般运输到实验室应尽可能的快。然而，如果不能避免更长的运输时间，运输在4°C的含氧解决方案可能是有利的。 1。 Prearrangements <ol><li>开关，它控制的的夹套烧杯中（ 表II）的温度上的thermocirculator。确保烧杯内的温度等于37℃玻璃盖盖住烧杯内的烧杯中，在整个隔离的过程中，以保持恒定的温度。 </li><li>称重胶原酶I和蛋白酶XXIV分为三个玻璃烧杯中，并在室温下存储。只是在使用前，将稀释的酶。 </li><li>专区20毫升的Ca 2 +在培养皿中的溶液在4℃下</li><li>存储250毫升Ca 2 +的溶液在37℃的水浴中</li></ol> 2。组织的清洁<ol><li>组织样品转移到陪替氏培养皿（直径约10厘米）的传输解决方案，连同大致用剪刀去除脂肪组织。 </li><li>称取组织样本。 200和600毫克之间，用于细胞分离。其它组织可以在液氮中冷冻后用生化分析。 </li><li>组织样本转移到培养皿中含有20毫升的Ca 2 +免费的解决方案，在4°C（见步骤1.3），切碎成约1毫米大小的小块组织样本。 </li><li>在37℃下连续脱气100％O 2，应执行以下步骤。枪头可以放置到氧气管，避免强烈的冒泡并产生持续的空气流。 </li><li>一起的Ca 2 +的溶液中转移的组织块夹套的烧杯中，小心搅拌约3分钟，使用磁性搅拌棒。 </li><li>停止搅拌，使组织块安顿下来了几秒钟。小心地应变的上清液，通过尼龙筛（200微米， 表II）。回到的内敛组织块从网格到烧杯中，使用产钳。 </li><li>笔芯烧杯与Ca 2 +免费的解决方案，重复洗涤步骤2.4和2.5的两倍。 </li></ol> 3。第一酶消化<ol><li>重新挂起的组织块用20毫升酶液含有胶原酶和蛋白酶E1（ 表一），并仔细搅拌10分钟。 </li><li>加入40μl的10mM 的 CaCl 2溶液，以获得最终浓度为20μM的Ca 2 +。 </li><li> 35分钟后仔细应变的上清液通过尼龙筛（200微米， 表Ⅱ）的方式，大部分组织块保持在烧杯中。回到休息下雨组织从网格块放入烧杯中。 </li></ol> 4。第二酶消化<ol><li>再次重悬的组织块，20毫升酶溶液E2含胶原酶我只（ 表一）。立即加入40μl的10mM 的 CaCl 2溶液，以获得最终浓度为20μM 的 Ca 2 +。 </li><li>在第二消化利用剪刀进一步砍组织的血块偶尔。 </li><li> 5分钟后，用巴斯德吸管的样品，以检查细胞的离解。重复此每隔2-3分钟，直到出现杆状，横纹肌心肌。 </li><li>停止搅拌，并允许组织块约20-30秒安顿下来。 </li><li>仔细应变上清到50毫升猎鹰管（管）的尼龙筛（200微米， 表二 ）。回到的内敛组织块从网格到烧杯中。 </li><li>重新暂停在烧杯中的组织块用20毫升斯道戈溶液中（ 表I）10，并进一步解离的细胞轻轻机械磨碎，用20毫升血清吸管与分配器。尽量避免形成泡沫，因为泡沫是不利于细胞的存活和质量。 </li><li>仔细应变上清到50毫升猎鹰管（B管）的尼龙筛（200微米， 表二 ）。 </li></ol> 5。最后准备和调整最终的Ca 2 +浓度<ol><li> 95 XG 10分钟，同时离心猎鹰管A和B（见步骤4.5和4.7）。 </li><li>从两个管仔细取出上清液愿望。确保不打扰颗粒。弃上清。 </li><li>将重新挂起既颗粒在1.5毫升的存储解决方案（ 表一），（室温）。 </li><li>加入7.5微升10毫米氯化钙溶液各猎鹰两次，仔细搅拌。孵育10分钟后，每一个步骤。 </li><li>加入15微升的10mM 的 CaCl 2溶液，以获得最终的Ca 2 +浓度为0.2 mM。 </li></ol> 6。肌细胞与荧光的Ca 2 + -指示器氟-3 AM（ 图1）中<ol><li> 1.5 ml的细胞悬液（管A和/或B管）转移到2 ml离心管（Eppendorf管）。 </li><li>正在考虑的光灵敏度的荧光的Ca 2 +指标氟3，应执行以下步骤。 </li><li>将50微克膜可渗透的乙酰氧基甲基酯衍生物氟-3（氟上午03点， 表III）溶解在44微升的Pluronic F-127（ 表III）储备溶液（20％w / v的在无水DMSO）得到1 mM的氟-3 AM原液，它可以储存在-20℃下，在1星期。 </li><li>加入15微升的萤光-3 AM原液含1.5 ml的细胞悬液（见6.1）和搅拌离心管仔细。 </li><li>孵育的细胞悬浮液，在不透光的框中，然后在10分钟。 </li><li>短暂离心在6000转左右。 </li><li>弃上清，重新悬浮颗粒在1.5毫升沐浴液（ 表四 ）。 </li><li>留下的细胞悬浮液约30分钟脱酯化，然后开始实验。 </li></ol> 7。同时膜片钳和外荧光的Ca 2 +测量由于膜片钳测量是这次审查的主要议题，是指有兴趣的读者提供更深入的描述，这种技术的其他出版物。11-14为了完整性，我们提供了一个简要的协议来衡量动作电位或L型Ca 2 +的电流，同时Ca 2 +的瞬态录音。 在实验过程中，心肌灌流在37°C洗澡的解决方案（ 表四 ）利用快速灌注系统（Octaflow IITM科学仪器，ALA，NY）。对于电压钳实验中，K +电流被阻塞，加入4 -氨基吡啶（5毫摩尔/升）和2（0.1 mmol / L的）氯化钡槽液。高硼硅玻璃微电极的使用和应该小费2-5MΩ电阻时充满吸管解决方案（ 表五 ）。除了 ​​氟-3 AM加载肌细胞（见步骤6），氟-3还包括电极内液（ 表V）。在488 nm处荧光激发和发射光（&lt;520纳米）转换的[Ca 2 +] i的假设<img alt="公式1" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> 其中，K D =解离常数萤光-3（864 NM），F =氟3荧光，F 最大 =的Ca 2 +-饱和每个实验结束时获得的荧光12这两个电信号和落射荧光的Ca 2 +信号被同时记录。动作电位刺激在1毫秒1.2倍阈强度的电流脉冲电流钳模式下使用的0.5赫兹。 L-型Ca 2 +的电流测量在电压钳模式下使用，保持电位为-80 mV快速失活的 Na +电流，其次是100毫秒100毫秒的斜坡脉冲到-40 mV在0.5 Hz至+10 mV的测试脉冲。

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N., 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=Different+compartments+of+sarcoplasmic+reticulum+participate+in+the+excitation-contraction+coupling+process+in+human+atrial+myocytes.">Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.</a> Circ. Res. 80, 345-353 (1997).</li><li>Heidbuchel, H., Vereecke, J., Carmeliet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+different+potassium+channels+in+human+atrium.+Contribution+to+the+basal+potassium+conductance.">Three different potassium channels in human atrium. Contribution to the basal potassium conductance.</a> Circ. Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., 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=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., 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=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

沃辛顿生化公司支持本出版物覆盖生产成本的视频文件。资助者没有参与研究设计，数据收集和分析，决定发表或准备手稿。

在这里，我们描述一个人的心房肌细胞从心脏直视手术的患者获得右心耳隔离方法。为了使用这些测量肌细胞胞浆钙2 +省略EGTA的存储解决方案，我们改编了前面描述的方法4-11。 早在1970年，它被观察到，虽然心肌细胞中存在的Ca 2 +在消化过程中分解，所有这些都是在挛缩和非可行。16,17因此，进行细胞分离，在Ca 2 +的解决方案。然而，重新引入生理浓度的Ca 2 +导致快速的Ca 2 +内流和细胞死亡。这已被描述为对Ca 2 +的悖论现象，它最初是灌注心脏中观察到的由Zimmerman和Hulsman 18修改的隔离介质的pH值下降到7.0，&lt;线上已经提出了防止Ca 2 +的悖论17&gt; 19此外，牛磺酸20或少量的Ca 2 +的（见步骤3.2和4.1），21，以及存储包含存储溶液22的离体心肌细胞中EGTA然而，这是众所周知的，Ca 2 +的缓冲通过EGTA降低的幅度的L-型Ca 2 +电流诱导的Ca 2 +的Ca 2 +瞬变的双相衰减的瞬时幅值和结果。23因此，我们省略EGTA在整个隔离为了获得具有典型属性和单相衰减的Ca 2 +瞬变过程。为了保护细胞的Ca 2 +的悖论我们最后的Ca 2 +浓度的存储解决方案以逐步的方式增加，直到为0.2mM。 胶原酶的选择可能是最关键的步骤，用于成功的细胞隔离。常规科尔agenases从溶组织梭菌得到的粗制剂和含有胶原酶除了一些其他的蛋白酶，polysaccharidases和脂酶。基于其一般组成胶原酶被分为不同的类型。24沃辛顿胶原酶类型I和II已经成功地用于人的心房肌细胞的隔离。4-10,15,25-30在我们目前所描述的协议，我们建议使用胶原酶I型，虽然我们还能够获得可接受的金额，使用胶原酶II型活菌。然而，即使在一个单一的胶原酶类型有一个显着的批次与批之间的变化有关的酶的活性。这些变化需要仔细的各种批次的批次选择和测试优化分离过程。网上可用的批次选择工具从Worthington生化股份有限公司（ <a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / CLS / match.php的）可用于查找可用批次的组合物，已被证明是适合于人类心房肌细胞的隔离。目前，我们使用I型胶原酶250 U /毫克胶原酶活性，345ü/毫克caseinase的活动，2.16 U /毫克梭菌的活性和0.48 U /毫克胰蛋白酶活性（批号＃49H11338）。 膜片钳研究，暂态Ca 2 +测量和另外两个15位的组合，也可以使用在8小时之内的使用本稿件所述的方法获得的细胞，这些细胞的允许响应电场刺激的细胞收缩的测量或电刺激用膜片钳移液器（未发表意见）。

心脏离子通道膜片钳技术和探索细胞内Ca 2 +处理异常需要隔离心肌的电生理特性研究。这些通常是在体外暴露心脏组织样品的消化酶（胶原酶，透明质酸酶，肽等）后得到的。大量的协议以来的首份报告于1955年2可行的心肌细胞的隔离已经开发以收获单心房和心室的心肌细胞，包括小鼠，大鼠，兔，狗，豚鼠和人类的不同物种。在这次审查中，我们侧重于人的心房肌细胞的隔离。关于隔离来自其他物种的心肌细胞，我们称之为“沃辛顿组织分解指南”沃辛顿生化公司，美国（ <a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ）提供程序。 等3所描述的方法，在这里，我们提供了一个一步一步的描述，这是改编自先前公布的方法，以一种技术获得心房肌细胞不仅适用于膜片钳实验，但也同时细胞内Ca 2 +的测量。4-11

<table border="1">
 <tbody>
 <tr>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td>Transport solution</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A3059</td>
 <td>Storage solution: 1%</td>
 </tr>
 <tr>
 <td>BDM</td>
 <td>Sigma-Aldrich</td>
 <td>31550</td>
 <td>Transport solution: 30</td>
 </tr>
 <tr>
 <td>DL-b-Hydroxy-butyric acid</td>
 <td>Sigma-Aldrich</td>
 <td>H6501</td>
 <td>Storage solution: 10</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Transport solution: 20 
 Ca2+-free solution: 20 
 Enzyme solution E1 and E2: 20 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>L-Glutamic acid</td>
 <td>Sigma-Aldrich</td>
 <td>G1251</td>
 <td>Storage solution: 70</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Transport solution: 10 
 Ca2+-free solution: 10 
 Enzyme solution E1 and E2: 10 
 Storage solution: 20</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td>Transport solution: 1.2 
 Ca2+-free solution: 1.2 
 Enzyme solution E1 and E2: 1.2 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich</td>
 <td>M9397</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Transport solution: 100 
 Ca2+-free solution: 100 
 Enzyme solution E1 and E2: 100</td>
 </tr>
 <tr>
 <td>Taurin</td>
 <td>Sigma-Aldrich</td>
 <td>86330</td>
 <td>Transport solution: 50 
 Ca2+-free solution: 50 
 Enzyme solution E1 and E2: 50 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>Collagenase I</td>
 <td>Worthington</td>
 <td>4196</td>
 <td>Enzyme solution E1 and E2: 286 U/ml</td>
 </tr>
 <tr>
 <td>Protease XXIV</td>
 <td>Sigma-Aldrich</td>
 <td>P8038</td>
 <td>Enzyme solution E1 and E2: 5 U/ml*</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 7.00 
 Ca2+-free solution: 7.00 
 Enzyme solution E1 and E2: 7.00 
 Storage solution: 7.40</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 1 M NaOH 
 Ca2+-free solution: 1 M NaOH 
 Enzyme solution E1 and E2: 1 M NaOH 
 Storage solution: 1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Concentrations in mM unless otherwise stated. BDM, 2,3-Butanedione monoxime. *Protease XXIV is included in Enzyme solution E1 only.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table I. Solutions.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon mesh (200 &mu;m)</td>
 <td>VWR-Germany</td>
 <td>510-9527</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Jacketed reaction beaker</td>
 <td>VWR</td>
 <td>KT317000-0050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table II. Specific equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl-sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-3 AM (special packaging)</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table III. Substances for loading of myocytes with Fluo-3 AM.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Bath solution</td>
 </tr>
 <tr>
 <td>4-aminopyridine*</td>
 <td>Sigma-Aldrich</td>
 <td>A78403</td>
 <td>Bath solution: 5</td>
 </tr>
 <tr>
 <td>BaCl2*</td>
 <td>Sigma-Aldrich</td>
 <td>342920</td>
 <td>Bath solution: 0.1</td>
 </tr>
 <tr>
 <td>CaCl2 &times; 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C5080</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Bath solution: 4</td>
 </tr>
 <tr>
 <td>MgCl &times; 6H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M0250</td>
 <td>Bath solution: 1</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Bath solution: 140</td>
 </tr>
 <tr>
 <td>Probenecid</td>
 <td>Sigma-Aldrich</td>
 <td>P8761</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 7.35</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 1 M HCl</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table IV. Bath solution for patch-clamp. *4-aminopyridine and BaCl were included for voltage-clamp experiments only.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Pipette solution</td>
 </tr>
 <tr>
 <td>DL-aspartat K+-salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2025</td>
 <td>Pipette solution: 92</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378</td>
 <td>Pipette solution: 0.02</td>
 </tr>
 <tr>
 <td>GTP-Tris</td>
 <td>Sigma-Aldrich</td>
 <td>G9002</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Pipette solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Pipette solution: 48</td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td>Pipette solution: 1</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td>Pipette solution: 4</td>
 </tr>
 <tr>
 <td>Fluo-3**</td>
 <td>Invitrogen</td>
 <td>F3715</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>7.20</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table V. Pipette solution for patch-clamp*. *On experimental days pipette solution is stored on ice until use. **Fluo-3 is added from a 1 mM stock solution on experimental days.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of human atrial myocytes for simultaneous measurements of ca2+ transients and membrane currents

研究心脏离子通道的电生理特性与膜片钳技术的探索心肌细胞内Ca 2 +处理异常需要隔离的心肌细胞。此外，调查使用这些技术从患者的心肌细胞，是一种宝贵的要求阐明的分子基础心脏疾病如房颤（AF）的可能性。1在这里，我们描述了一个隔离的人的心房肌细胞的方法既适合膜片钳研究和细胞内Ca 2 +浓度的同时测量。首先，从心脏直视手术的患者获得右心耳切成小组织块（块法“），并在Ca 2 +免费溶液洗涤。然后在含有20μM的Ca 2 +的解决方案与胶原酶和蛋白酶消化组织块。此后，将离体心肌细胞收割机受激发射损耗的组织悬浮液的过滤和离心。最后，Ca 2 +浓度在单元格中的存储解决方案的逐步调整，以0.2mM的。我们简要的Ca 2 +和Ca 2 +缓冲隔离过程中，还提供有代表性的录音，动作电位，膜电流，两者一起同时暂态Ca 2 +的测量，进行离体心肌细胞在这些讨论的意义。

图2A显示了三个有代表性的例子，从孤立的人右心房肌细胞。为了量化我们移取10微升的细胞悬液（5.5）上CellFinder显微镜幻灯片（ <a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.html</a> ）的细胞产量。平均细胞产量图2B清楚地表明，有一种倾向，以降低细胞产量在慢性房颤（CAF）患者样本（管答：16.5±3.1 cells/10微升组（n = 29）与5.1±2.3 cells/10微升组（n = 10）在SR和咖啡馆，分别为，P &lt;0.05;管B：17.9±3.9 cells/10微升组（n = 29）与5.9±2.0 cells/10微升SR和CAF组（n = 9）， ，P = 0.107）。 动作电位的测量和同时录音的胞浆内的Ca 2 +瞬变的代表性的例子在图3中给出。在约90％的所研究的细胞，吨他动作电位触发的Ca 2 +释放引起明确和正常细胞收缩。如先前报道，静息膜电位，细胞的完整性，这是一个公认的指标，平均约-73.9±2.7 mV的（N = 23/10肌/例）和-77.7±1.8 mV的（N = 19/8肌/病人15）SR和CAF（P&gt; 0.05）。 图4显示了电压门控L-型Ca 2 +电流和胞浆内Ca 2 +的瞬变代表同时录音。非选择性β-肾上腺素能受体激动剂异丙肾上腺素（1μM）中的应用两个I 钙，L和胞浆中的Ca 2 +瞬变的幅度增加，提示完整的β-肾上腺素能的信号转导级联反应。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> FIGURE 1。流程图的心肌细胞萤光-3 AM加载协议（见步骤6.1-6.5）。 M / V，质量/体积。 <img alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> 图2。 A，隔离人类右心房肌细胞存储解决方案，一小时后，B，平均值±标准差在10微升的存储解决方案中的细胞计数细胞产量（见第5.5步）。 n指的数量各组内的准备。 * P &lt;0.05。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> 图3。动作电位触发的Ca 2 +瞬变（CAT）从窦性心律心房肌细胞和字符代表录音ONIC房颤病人。顶：注入膜电流（I M）用于刺激（0.5赫兹）。如下：同时记录膜电位（V M），并引发猫（下）。 （重绘许可从福格特等人。2012）15 <img alt="图4" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> 图4。代表录音的异丙肾上腺素（1μM）的效果，在L-型Ca 2 +电流触发的Ca 2 +瞬变（CAT）从窦性节律和慢性心房颤动患者的心房肌细胞。顶：电压钳位协议（0.5赫兹）。如下：同时记录总净膜电流（I M），主要反映了L-型Ca 2 +电流（中）和触发猫（下）。 （重绘许可福格特，等，2012）15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="表1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> 表一解决方案。 <img src="/files/ftp_upload/50235/50235table2.jpg" alt="表2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> 表II中。具体设备。 <img src="/files/ftp_upload/50235/50235table3.jpg" alt="表3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> 表III。物质加载萤光-3肌AM。 <img src="/files/ftp_upload/50235/50235table4.jpg" alt="表4" fo:conteNT宽度=“2.5英寸”FO：SRC =“/ files/ftp_upload/50235/50235table4highres.jpg”/&gt; 表四。膜片钳的沐浴解决方案。 <img src="/files/ftp_upload/50235/50235table5.jpg" alt="表5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> 表五液膜片钳*。

Watch this Scientific Journal Video about Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents at JoVE.com

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

我们描述了人类心房肌细胞可用于细胞内Ca隔离<sup&gt; +</sup&gt;组合测量膜片钳电生理研究。

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

isolation-human-atrial-myocytes-for-simultaneous-measurements-ca2

Research

JoVE Journal

Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

20.2K 次观看.  University of Duisburg-Essen. 我们描述了人类心房肌细胞可用于细胞内Ca隔离 +组合测量膜片钳电生理研究。

视频: Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

成人的心肌细胞的分离和遗传操作共聚焦成像

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

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 ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

在小鼠的心房和心室肌细胞的分离及钾离子通道的录音

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...