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?...

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		<th>Material Name</th>
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<td>Anesthesia solution</td>
<td>&nbsp;</td>
<td>Serumwerk Bernburg GmbH (Ursotamin), Bayer Health Care (Rompun)</td>
<td>&nbsp;</td>
<td>Mixture of 1 ml Ursotamin (100 mg/ml Ketaminhydrochlorid) and 240 &mu;l Rompun (2% Xylazinhydrochlorid)</td>
</tr>
<tr>
<td>Citrate solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>117,64 mg sodium citrate in 10 ml 0.9% NaCl solution</td>
</tr>
<tr>
<td>Solution A</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>134 mM NaCl, 4 mM KCl, 11 mM glucose, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Solution A+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 0.2 mM EGTA, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
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<td>Liberase solution</td>
<td>&nbsp;</td>
<td>F. Hoffmann&#x2010; La Roche Ltd.</td>
<td>11988476 001</td>
<td>500 &mu;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>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 200 &mu;M CaCl2 and 0.1% DNase solution</td>
</tr>
<tr>
<td>Solution B1/2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1:1 mixture of solution A and solution B</td>
</tr>
<tr>
<td>DNase solution</td>
<td>&nbsp;</td>
<td>Sigma&#x2010; Aldrich Co.</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&#x2010;HCl buffer, adjusted to pH=7.35, additional content: 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol; this solution is mixed with 5 ml glycerol</td>
</tr>
<tr>
<td>Extracellular matrix protein (ECM) solution</td>
<td>&nbsp;</td>
<td>Extracellular matrix protein (ECM) solution</td>
<td>E1270</td>
<td>1 ml stock solution as purchased is diluted with 6 mL
medium M199</td>
</tr>
<tr>
<td>Culture medium M199</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>E15&#x2010;834</td>
<td>Medium M199 supplemented with 1 &mu;l/ml ITS solution and 20 &mu;l/ml penicillin&#x2010;streptomycin solution (5000 units/ml)</td>
</tr>
<tr>
<td>ITS solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>25 mg Insulin, 25 mg Transferrin and 50 &#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 clear
solution. Aliquots can be frozen.</td>
</tr>
<tr>
<td>Tyrode</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 2mM MgCl2, 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>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5 &mu;l 10&#215;PCR&#x2010;Buffer, 5 &mu;l MgCl2 (25 mM), 2 &mu;l forward primer, 2 &mu;l reverse primer, 1 &mu;l dNTP&#180;s (10 mM each), 33 &mu;l H2O, 1 &mu;l DMSO, 1 &mu;l Taq&#x2010;DNA polymerase for each sample</td>
</tr>
<tr>
<td>LB&#x2010;Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</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>&nbsp;</td>
<td>New England Biolabs</td>
<td>R0547L</td>
<td>preparation: 50 &mu;g DNA, 5 &mu;l 10&#215;buffer, 0.5 &mu;l 100&#215;BSA,
0.75 &mu;l Pac I, fill with H2O up to final volume of 50&mu;l</td>
</tr>		</tbody>
		</table>
		</div>

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

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

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

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

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

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+ ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

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 ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

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). ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...