这项工作是由德国国家科学基金会（DFG）和联邦风险评估研究所（BFR，德国）的支持。

对所有的协议，包含四个主要步骤的设计如图1所示。所有步骤都在全面详细描述。细胞分离，转导和文化，由大约24个小时的共焦成像后是一个连续的和强制性的时间表。该病毒的建设应该做超前的细胞的分离和基因转导，然后使用。 1。成年大鼠心脏心肌细胞的分离<ol><li>成年雄性Wistar大鼠重量约250克（6-12周龄）腹腔注射170μL麻醉每100克体重的解决方案anesthetised </li><li>此外，柠檬酸钠溶液（2μL/ g体重）注射，以避免手术过程中血液凝固。 </li><li> Langendorff灌流的设置应准备。所有的解决方案应与氧饱和。 </li><li>当老鼠是anesthetised，动物固定​​在手术板上和身体的上半部分是用70％乙醇消毒。 </li><li>通过切割颈动脉的动物被杀害。 </li><li>胸部是用剪刀打开。心脏和主动脉现在应该访问。 pericard被删除。 </li><li>冰冷溶液A +（5毫升）脑室注射均使用26号针头逮捕的心脏和洗出的血液。 </li><li>主动脉抓着钳截断主动脉弓的第一个分支发生。 </li><li>从胸部心脏和主动脉连接到由钳灌注设置和钳位套管，允许逆行的Langendorff灌注。请注意，不要foraminate的主动脉瓣。 </li><li>心脏需要围绕主动脉/导管结扎固定。开放的船只也需要予以结扎。 </li><li>逆行灌注心脏是与氧饱和溶液在室温下约70毫巴的压力在10分钟（23℃）+。确保无气泡进入主动脉。 </li><li>灌注液温度在37到一个Liberase解决方案交换℃，约25分钟蠕动泵率在4毫升/分钟。在灌注结束的心应该寻找大理石白色。消化时间可能需要适应形势/组织条件。 </li><li>取自心脏灌注设置，并在培养皿中的解决方案包含一个放置在温度37 ° C剩余的船只和心房被切断。这些船只将被丢弃和心房 - 如果需要的话 - 区别对待（不是这个协议的一部分）。心室分为约6件。 </li><li>件转移到100毫升锥形含有20毫升溶液在温度37℃，并在37℃水浴5分钟稍微激动。烧瓶最后被丢弃上清液。 </li><li>上述过程重复一次或两次。上清应该出现轻微不透明。 </li><li>开始添加20毫升溶液B 1 / 2，在37 ° C到细胞和组织，其余的解决方案，剩余的胞外钙离子浓度的增加是一个锥形瓶中。这是有点动摇了，37℃水浴一个额外的5分钟。随后被丢弃上清液。 </li><li>进一步增加20毫升溶液B：1 / 2 。 （解决方案/上清现在应该包含活菌）。细胞下降是一个倒置细胞培养显微镜转移到培养皿。如果细胞显示出较高的自发活动（收缩）之一需要等到细胞来休息。 </li><li>再一次被丢弃上清液，并添加20毫升溶液B 1 / 2 。 </li><li>以这样的方式，组织块时，容易激动，通过开放，可以通过塑料巴斯德吸管尖被切断。具有阻燃处理，锐利的边缘软化（融化）。 </li><li>现在的组织块稍微轻轻磨碎。组织块“土崩瓦解”，进入细胞内。 </li><li>细胞含有上清倒出，并与B液稀释，以达到所需的细胞密度，这取决于类型的实验，并应凭经验确定。 </li></ol> 2。建筑的融合蛋白转导腺病毒生产腺病毒的移调，广告MP生物医学的腺病毒载体系统是用 1 。该协议开始与现有的和测试向量入口为荧光融合蛋白或创业板第2条 （pCR259）。 <ol><li>上述初步pC​​R259包含感兴趣的基因的载体的10纳克量100μLHighQ移调- AD - 1™294胜​​任细胞转化。 </li><li>这种混合物孵育20分钟冰热休克45秒在42 ° C，其次是存储在一个额外的2分钟冰。 </li><li>新增900＆万亩;升非选择性LB ​​培养基和成长在37 ° C的4小时轻轻摇动。 </li><li>板50μL，100μL和200μL含有氯霉素（CM，20微克/毫升），氨苄青霉素（100微克/毫升），四环素（15微克/毫升），X - Gal的（280微克/毫升LB培养基板）和IPTG（40微克/毫升）。 （可能是一个更加激烈和更快的蓝染色300μg/ml使用较昂贵的染料Bluo - Gal的，这应该是尤其当你没有识别和区分蓝色/白色菌落如此熟悉） </li><li> 24小时，增长在37 ° C。在4℃直至离开良好的识别蓝色菌落可能。当有问题，以决定它的殖民地，是真正的白色挑选的一些殖民地，并复制在另一盘含有三种抗生素，X - gal和IPTG，让他们成长在夜间在37 ° C. 备注）HighQ - 1移调AD™294细胞中含有两个重要的质粒，腺病毒骨架（移调- AD™294）和辅助质粒表达了一个感兴趣的基因插入到它的目标网站，从而介导的转座，位于在移调公元294 LacZ基因质粒。 </li><li>要确定阳性克隆含有基因的病毒骨干的利益进行菌落PCR在2.7-2.9所述。使用载体引物侧翼多个克隆网站（向前5&#39;CCCAGTCACGACGTTGTAAAACG 3&#39;，反向5&#39;GCGGATAACAATTTCACACAGG 3&#39;），并与100％的白色菌落菌落PCR和使用无插入的基因作为控制移调-公元294质粒一个蓝色的殖民地利益。蓝色菌落会给你一个简短的PCR片段（〜200bp），包含您感兴趣的基因的殖民地，一个大的PCR产物（取决于你感兴趣的基因的长度），与两个片段多个细胞克隆菌落，必须分开如果进一步使用。 </li><li>根据下面的主结构表，需要有所准备。 </li><li>蒸压牙签挑选一个100％的白色菌落，首先要对这个殖民地复制与厘米的LB板。 </li><li>转移到PCR混合殖民地。在PCR混合捻取出牙签之前，可以浸泡很多的PCR混合的3-4倍。 PCR协议取决于插入和使用聚合酶KB。引物的退火温度是57 ° C和35个循环使用建议。 </li><li>增长的阳性克隆的夜间文化（LB培养基与CM）和隔离与商业没有离心柱的试剂盒质粒，因为，在离心力的作用，破坏了庞大的病毒载体。 </li><li>变换0.5微克100μL的DH5α感受态细胞中分离出质粒。孵育30分钟冰细胞的DNA。热休克45秒42 ° C和冰浴2分钟的设置。 </li><li>加入900μL无选择性LB培养基，生长在37℃，轻轻摇动1小时。 </li><li>板50μL含厘米，X - gal和IPTG的LB培养基上板。在37 ° C培养24小时。 </li><li>选择和复制20 100％单独氨苄青霉素，四环素和氯霉素板含有IPTG和X - Gal的白色菌落。让他们在一夜之间成长在37 ° C。选择是100％的白色，耐氯霉素，氨苄青霉素和四环素敏感的殖民地。 </li><li>菌落PCR（可选）：阳性克隆可与殖民地PCR测试，检查它们是否包含插入。 </li><li>挑选单菌落了积极的重组，并与CM 600毫升LB培养基中生长。 </li><li>使用质粒马克西试剂盒分离的质粒。 </li><li>精华5微克质粒用Pac我的两个小时，在37 ° C和反应停止在65 ° C为20分钟。 </li><li>在Speed​​Vac整个消化集中在室温为30分钟到终体积为10μl，或使用酚 - 氯仿的提取方法。 </li><li>解冻一小瓶冷冻QBI - HEK 293细胞在37℃水浴中轻轻摇动。小瓶用70％乙醇冲洗外，并在无菌罩在无菌条件下进行。 </li><li>传递细胞15毫升无菌试管中，滴加10毫升的DMEM（10％FCS）的添加而温柔的搅拌，并在250xg离心10 min沉淀细胞。 </li><li>弃上清。于10 ml DMEM（10％FCS）的向上和向下轻轻吹打重悬沉淀。 </li><li>种子在75厘米2烧瓶，添加10毫升含10％FCS的DMEM细胞。 37 °，在夜间在5％的CO 2培养箱彗星。 </li><li>第二天验证的细胞密度。 QBI - HEK 293细胞，应分别在1:10至1:20的比例。 </li><li>从细胞中取出的中型和trypsinize 2毫升为1-3分钟胰蛋白酶。给予10毫升的DMEM细胞，并在250xg离心10分钟。 </li><li>小心取出介质和细胞10毫升的新鲜培养基，重新悬浮细胞计数。 </li><li>板六以及与5 2X10，3X10 5 </s&gt; 4 × 5，5X10 5和6x10 5个细胞。菜在37 ° C中的CO 2培养箱。 </li><li>使用50％汇合在翌日良好的转染与集中的PAC我消化。我们有很好的经验与脂质体2000转，但还有其他方法（如磷酸钙转）应该是合适的。 </li><li>在转染前3-4小时更改中期进入指数增长，在手术过程中的细胞。 </li><li> 5μL脂质体在室温下混合5分钟与250μLDMEM培养。 </li><li>给予脂质体中等混合的DNA“（PAC我消化），并通过对反应管，混合，不吹打混合。在室温下孵育混合物20分钟。 </li><li>掉落细​​胞的转染混合物和岩石混合板。在37 ° C孵育5％的CO 2。更改后4-5个小时的中等。 </li><li>转染后两天从细胞中，并给到一个新的培养瓶（75厘米）。 </li><li> Trypsinize用500μl胰酶长达5分钟的细胞。给予1毫升的DMEM细胞，整个混合物，并把它连同17毫升新鲜培养基的培养瓶中（与FCS的DMEM）。培养的细胞在37℃，5％的CO 2，直到细胞病变效应（CPE），是可见的。见图2，学习如何CPE应该像。 </li><li>与介质的细胞收集在50毫升管和冻结的细胞在-80 ° C。打破三个冻结/解冻循环（-80 ​​° C/37 ° C水浴）的细胞。 </li><li>离心20分钟，在8000xg和4 ° C。到一个100mm直径碟倒出上清液，弃去沉淀。使用孔径0.45μm的无菌过滤，滤液上清。这是第一个病毒的股票（通道1）。 </li><li>使用这种股票的1 / 3转导一个75厘米 2烧瓶QBI - HEK 293细胞70％汇合。在37 ° C和5％的CO 2，直到CPE的小号是可见的。 </li><li>如果细胞CPE的小号收集和提取的病毒之前完成。这是通过病毒2。 </li><li> 2.37和2.38病毒通过重复步骤2和病毒的扩增，依此类推，直至达到足够的病毒库存。该病毒可集中与特定的离心管（Amicon超15离心ultracel - 100微孔膜过滤装置），数个月，储存在-80 ° C要计数的传染性颗粒，使用斑块检测。 </li><li>通过2或更高的病毒转导的细胞和病毒的进一步放大。 </li></ol> 3。原代细胞培养和腺病毒转导<ol><li>无菌直径20毫米的圆形盖玻片，放置在标准12孔板。 </li><li> 20μL的ECM解决方案是均匀分布在每个盖玻片。遗体被丢弃。让干燥至少1小时在37 °或者涂层可以通过干燥，夜间在室温（12小时）。 </li><li>每口井是充满了1毫升中等M199包括抗生素和ITS的补充。 </li><li> （从1.21步）一个200至400μL细胞悬液，每孔加入。培养的细胞发生在37 °彗星在5％ 二氧化碳培养箱。 </li><li>细胞允许泥沙和1 h后的介质改变。 </li><li>一个足够量的病毒解决方案（通过2或更高）混合后立即改变中期轻摇板。 </li><li>在一个长期培养基的情况下，每2天。 </li><li> 24小时后转创业板的表达被觉察。 </li></ol> 4。共焦成像3D的亚细胞结构和Ca 2 +信号可以执行任何共聚焦显微镜共焦成像。然而，钙离子成像实验需要至少视频率（25-30赫兹）的采集速度。这就限制了可用的技术或者多束扫描仪（如尼普科夫光盘，席卷领域或公斤光束阵列扫描仪）或非常快的单光束扫描仪（如共振扫描仪或声光偏转器（AOD），扫描仪）。有关概述，见参考文献。 3。亚细胞的三维成像，需要一个Z驱动器。在这里，一公斤光束阵列扫描仪（VTinfinity，VisiTech诠释，桑德兰，英国）使用。 <ol><li>显微镜及周边设备需要设置到运作模式。特别是激光，这可能需要变暖期，直到达到一个稳定的输出功率。 </li><li>细胞需要被转移到一个试验室（盖滑转移）。与细胞的盖玻片被转移到一个试验室。然后加入1ML Tyrode液（含1.8毫米的细胞外钙）。 </li><li>试验室需要放置在显微镜舞台上。刺激电极，灌注设置和进一步peripheRAL设备，如温度控制，需要作出相应安排。 </li><li>细胞将集中在使用白光和/或啶照明选择。 </li><li>采集参数，如激光功率，曝光时间等，或一个完整的实验协议需要设置。 </li><li>时间序列和/或Z节的图像可以被收购。 </li><li>在进一步设置先进的多模测量，可以使用电（膜片钳）和/或光学操纵像再分配后，荧光光漂白（FRAP）包括两个光子光解物质uncaging。 </li><li>图像分析，特别是如Imaris（位平面AG，瑞士苏黎世），速度（即兴创作，英国考文垂）或Arivis浏览器（arivis有限责任公司，德国罗斯托克）的专用分析软件进行三维重建。 </li></ol> 5。代表性的成果： 该协议的最终结果是图像，可用于提取进一步的数据序列。 一个例子是，亚细胞结构和细胞器的调查。图3描绘了高尔基体的3维排列的例子。所有的调查，在电子显微镜，可以进行活体细胞。 <img src="/files/ftp_upload/1433/JoVEFig1.jpg" alt="图1" /> 图1：一般流通的主要实验步骤。 <img src="/files/ftp_upload/1433/JoVEFig2.jpg" alt="图2" /> 图2：左图描绘文化的Q - HEK 90-95％汇合的细胞是不转。 Cytopatic病毒转导后的效果显示在右边的部分。细胞轮和分离板的底部。在这些细胞中的细胞核占据细胞的重要组成部分，作为一个活跃的病毒生产。 <img src="/files/ftp_upload/1433/JoVEFig3.jpg" alt="图3" /> 图3：成人与YFP的融合蛋白，针对高尔基体转染大鼠心肌细胞。腺病毒介导的基因转移（文化一天）后的心肌细胞表达的荧光融合蛋白。 A组描绘了一个单一的焦片从Z - Stack的。在B组中，描述了相同的细胞后，用一个理论点扩散函数（PSF）反卷积。随后用此去模糊图像的堆栈来呈现细胞和面板C或三维表面重建（四）所示，得到一个三维容积重建。酒吧代表10微米。

<ol>
	<li>Kaestner, L., Ruppenthal, S., S, ., Licha, K., Lin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Imaging II. Vol. 7370, 737008-737008 (2009).</li><li>Viero, C., Kraushaar, U., Ruppenthal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Cell Calcium. 43 (1), 59-59 (2008).</li><li>Kaestner, L., Lipp, P., Shorte, S. L., Frischknecht, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Cellular and Molecular Biological Functions. , 289-289 (2007).</li><li>Kaestner, L., Lipp, P., Popp, J., von Bally, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optics in Life Science. Vol. 6633, 66330K-66330K (2007).</li><li>Rinne, A., Littwitz, C., Bender, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+delivery+of+short+hairpin+RNA+(shRNA)+mediates+efficient+gene+silencing+in+terminally+differentiated+cardiac+myocytes.">Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.</a> Methods Mol Biol. 515, 107-107 (2009).</li></ol>

鼠心肌细胞的分离描述的过程可以适应其他物种，例如鼠标 4，如有必要。需要适应的一个参数，对消化酶的组合（见下文）和消化的时间。要知道，有些种类（如鼠标）的细胞可能会比其他人（如鼠）更脆弱。 胶原酶的选择可能是在隔离过程中最关键的一步。我们选择Liberase Blendzyme，因为它是一种合成酶的组合几乎没有批到批的变化。传统的方法是使用所谓的原油?...

<table><tbody><tr><td>Anesthesia solution</td><td>Serumwerk Bernburg GmbH (Ursotamin), Bayer Health Care (Rompun)</td><td></td><td>Mixture of 1 ml Ursotamin (100 mg/ml Ketaminhydrochlorid) and 240 &amp;#x03BC;l Rompun (2% Xylazinhydrochlorid)</td></tr><tr><td>Citrate solution</td><td></td><td></td><td>117,64 mg sodium citrate in 10 ml 0.9% NaCl solution</td></tr><tr><td>Solution A</td><td></td><td></td><td>134 mM NaCl, 4 mM KCl, 11 mM glucose, 1.2 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 1.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td></tr><tr><td>Solution A+</td><td></td><td></td><td>Content of solution A plus 0.2 mM EGTA, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td></tr><tr><td>Liberase solution</td><td>F. Hoffmann&amp;#8208; La Roche Ltd.</td><td>11988476 001</td><td>500 &amp;#x03BC;l stock solution in 15 ml solution A (stock solution:10 mg/ml Liberase Blendzyme 4 in aqua dest.)</td></tr><tr><td>Solution B</td><td></td><td></td><td>Content of solution A plus 200 &amp;#x03BC;M CaCl&lt;sub&gt;2&lt;/sub&gt; and 0.1% DNase solution</td></tr><tr><td>Solution B&lt;sub&gt;1/2&lt;/sub&gt;</td><td></td><td></td><td>1:1 mixture of solution A and solution B</td></tr><tr><td>DNase solution</td><td>Sigma-Aldrich</td><td>D4527</td><td>Deoxyribonuclease I, Type 2 from bovine pancreas, 40 KU (15 mg) are dissolved in 5 ml of 10 mM Tris&amp;#8208;HCl buffer, adjusted to pH=7.35, additional content: 50 mM NaCl, 10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM dithi&amp;#8211;rythritol; this solution is mixed with 5 ml glycerol</td></tr><tr><td>Extracellular matrix protein (ECM) solution</td><td>Extracellular matrix protein (ECM) solution</td><td>E1270</td><td>1 ml stock solution as purchased is diluted with 6 mLmedium M199</td></tr><tr><td>Culture medium M199</td><td>PAA Laboratories</td><td>E15&amp;#8208;834</td><td>Medium M199 supplemented with 1 &amp;#x03BC;l/ml ITS solution and 20 &amp;#x03BC;l/ml penicillin&amp;#8208;streptomycin solution (5000 units/ml)</td></tr><tr><td>ITS solution</td><td></td><td></td><td>25 mg Insulin, 25 mg Transferrin and 50 &amp;#x03BC;l Selenite stock solution are dissolved in 5 ml aqua dest. (stock solution: 0.5 mg/ml Sodiumselenite in aqua dest.). Add a few drops of 1 M HCl until one should get a clearsolution. Aliquots can be frozen.</td></tr><tr><td>Tyrode</td><td></td><td></td><td>135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 2mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td></tr><tr><td>Master mix</td><td></td><td></td><td>5 &amp;#x03BC;l 10&amp;#215;PCR&amp;#8208;Buffer, 5 &amp;#x03BC;l MgCl&lt;sub&gt;2&lt;/sub&gt; (25 mM), 2 &amp;#x03BC;l forward primer, 2 &amp;#x03BC;l reverse primer, 1 &amp;#x03BC;l dNTP&amp;#180;s (10 mM each), 33 &amp;#x03BC;l H&lt;sub&gt;2&lt;/sub&gt;O, 1 &amp;#x03BC;l DMSO, 1 &amp;#x03BC;l Taq&amp;#8208;DNA polymerase for each sample</td></tr><tr><td>LB&amp;#8208;Medium</td><td></td><td></td><td>10 g/l Tryptone, 5 g/l Yeast extract, 5 g/l NaCl (and 15 g/l Agar if used as solid medium), pH 7.0</td></tr><tr><td>Pac I</td><td>New England Biolabs</td><td>R0547L</td><td>preparation: 50 &amp;#x03BC;g DNA, 5 &amp;#x03BC;l 10&amp;#215;buffer, 0.5 &amp;#x03BC;l 100&amp;#215;BSA,0.75 &amp;#x03BC;l Pac I, fill with H&lt;sub&gt;2&lt;/sub&gt;O up to final volume of 50&amp;#x03BC;l</td></tr></tbody></table>

isolation and genetic manipulation of adult cardiac myocytes for confocal imaging

从成人心分离的心肌细胞模型某处一侧的胚胎和新生儿的肌肉细胞和其他工作的心脏之间的半路上被广泛接受。因此，心肌细胞，心脏细胞的生理和病理生理的良好模型服务，用于制药的调查，以及转基因动物模型的探索。在这里，我们描述了一种发自内心的细胞分离方法。此外，我们还展示了如何对心肌细胞的基因操纵的培育转基因动物可以不执行：这是长期文化的结合（1周）和腺病毒介导的基因转移。后者是描述，从病毒的建设转导的细胞。它可用于基因编码的生物传感器（GEBs），荧光融合蛋白的表达，同时也为蛋白质的过度表达和向下调节，如利用RNAi。在这里，我们提供一个融合的一个亚细胞结构（高尔基）染色的蛋白质表达例子。这种蛋白的表达，可以通过Z -堆叠的共焦成像的亚细胞结构的三维重建可视化。该协议包括在国家的最先进的细胞培养，分子生物学和生物物理学，从而提供了一个探索新的视野，在蜂窝心脏病的方法。

Watch this Scientific Journal Video about Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging at JoVE.com

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞原代细胞，可以从动物心脏中分离和培养几天。在这个文化时期的腺病毒介导的基因转移，可以用来表达基因编码的生物传感器（GEBs）或荧光融合蛋白。这两种方法都可以通过共聚焦显微镜细胞调查。

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 ...

isolation-genetic-manipulation-adult-cardiac-myocytes-for-confocal

Research

JoVE Journal

Biology

14.5K 次观看.  Universty of Saarland. 成人的心肌细胞原代细胞，可以从动物心脏中分离和培养几天。在这个文化时期的腺病毒介导的基因转移，可以用来表达基因编码的生物传感器（GEBs）或荧光融合蛋白。这两种方法都可以通过共聚焦显微镜细胞调查。

视频: Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

分离的月毛心室肌细胞的光学成像

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

科研

生物工程

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

生物学

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

医学

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

从固定心脏分离心肌细胞进行免疫细胞化学和倍体分析

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...