1。心肌细胞分离 材料（图1） Microdissecting钳 组织钳 细腻的止血钳 止血钳 Microdissecting，锯齿，弯钳 经营剪刀，直 经营剪刀，弯 15毫升Falcon管（5） 60毫米培养皿 磷酸盐缓冲盐水（PBS）中 尼龙网 - 400微米孔径 小漏斗 涂蜡，编织丝绸4-0 19毫米，长7-10厘米 非注射针，钝端，24 G或商业动物喂养针（24×1，W/1-1/4） 肝素钠 氯胺酮/甲苯​​噻嗪混合 巴斯德移液器 OPTIVISION解剖镜 IonOptix MMSYS系统 注意：如果培养细胞，见细胞培养第2节开始本协议之前。 注：所有小鼠均在照顾根据IACUC批准的法规，惯例和程序的障碍设施和牺牲。 注：在开始的过程中，整个系统必须进行预热，以确保Langendorff灌注系统（ 图2A，图3）的所有元素都被加热至37℃，以便正确和完整的胶原酶活性。 <ol><li>注入成年鼠（〜12周）与150 USP单位肝素钠进入腹腔空间。 </li><li>麻醉用在一个重量依赖性剂量（ 表1）注射氯胺酮/赛拉嗪的混合物。 <ol><li> 80:12毫克/千克氯胺酮：甲苯噻嗪配方：2.6毫升氯胺酮（100毫克/毫升）+0.4毫升甲苯噻嗪（100毫克/毫升）+ 37毫升的PBS。决赛：氯胺酮= 6.5毫克/毫升;甲苯噻嗪= 1毫克/毫升</li></ol></li><li>固定镇静鼠标操作垫，切除心脏，并放入室温的PBS的培养皿或0.9％生理盐水（ 图1J）。 PHYsiologic生理盐水允许完整心脏收缩在室内更好的挤出血液和易于识别步骤1.6之前的主动脉。小鼠进行了检查，以确保它们通过深入的后肢脚趾捏反射和呼吸频率发病前开胸的放缓损失麻醉。 </li><li>使用OPTIVISION解剖镜（ 图1A）来识别和揭露主动脉。除去主动脉周围的组织，虽然它确实有利于容器的可视化，是没有必要的细胞分离的优化，如果在主动脉根部清晰可见。 </li><li>素与灌注缓冲泵（ 表2）。温度应在37℃下使用保持的程序的长度的控制，循环水浴（ 图2D）。 </li><li>安装心脏到Langendorff装置上（ 图2A）。使用两个微解剖钳（ 图1D-E），PRIME主动脉周围无皮下，钝端针（24 G）与硅胶管的远端。可选地商业动物喂养针（24×1中，W/1-1/4）都可以使用。上的24号针头或动物喂食针的硅胶管将允许用于固定缝合线在槽。将主动脉上的针为一只袜子。 （ 图2C）。 <ol><li>注意：为确保针的端部搁置在升主动脉中，最好是在主动脉根部远侧至右无名是很重要的，但是，它并没有通过主动脉瓣进入左心室延伸，因为这会造成严重的限制缓冲器通过心脏通过冠状动脉有效灌注的能力。 </li></ol></li><li>通过把一个小的长度缝合丝（7-10厘米长）（ 图1K）主动脉周围的顶部，确保领带是在升主动脉的水平，低于右无名阿特保护心脏的针Y，但是是在针的末端上。 </li><li>降低心脏成锥形玻璃的内部（ 图2B），以帮助保持一个合适的环境温度。 </li><li>用5分钟的灌注缓冲液灌注心脏以1毫升/分钟的速率和夹持在玻璃腔室流出，以允许灌注液收集在锥形玻璃和包络的心脏。的示意图用于消化心脏的示于图3。 <ol><li>此时你应该看到的心脏增大音量。此外，血液在心脏组织会通过灌流液进行更换，造成组织苍白。 </li></ol></li><li>松开夹子流出排灌流。停泵从灌注缓冲容器将管线与酶缓冲容器（ 图2E）。重新启动泵，开始以1毫升/分钟，酶缓冲液（ 表2）灌注心脏。再用镊子流出，使酶缓冲的信封心脏。 <ol><li>这里，作为酶灌注心脏和消化结缔组织，心脏会变得苍白和结构的完整性会减弱。 </li><li>确定何时从消化心脏切开心室，解除心脏出了锥形玻璃，轻轻地用钳子挤压心脏，并收集了几滴灌流液中的小培养皿中，并检查单个细胞是否被丢弃。 </li><li>对于新手来说是有帮助的连接的压力计的灌注管线。当心脏已经消化压力将迅速下降，如结缔组织不成立的阻力。压力计也对新手来说非常有用，以确保针的位置是上述主动脉瓣，而不是在心室。低压将表明，该针是在心室腔和高压表明针触到阀。 </li></ol></li><li>切从心脏的心室成菜满转印缓冲液（A）（ 表2）当心室压力开始急剧下降，或当你能够看到单个细胞的灌流液。这通常需要〜7-10分钟。 </li><li>再次使用微解剖钳（ 图1C），剁碎心室切成小块解离。一个成功的消化将离开组织几乎没有固体块切碎后，与大多数组织成为解离后，非晶。 <ol><li>以进一步分离的组织，采用了塑料巴斯德吸管（ 图4），从该尖端已切于一个〜45°角度吸取进/出该溶液中。这种修改是可以增加的枪头从而减少对细胞的剪切应力的量为组织被研磨内径的空间。 </li><li>前解剖组​​织的钳子，可以分开单独的腔室，并分别分离心房，左心室或右击吨心室肌细胞。 </li></ol></li><li>固定平方目（ 图1M）的一个小漏斗（ 图1L）使用卡，并把这种过滤装置为15毫升猎鹰管（ 图1I）。 </li><li>使用修改过的巴斯德移液管从培养皿取出的细胞溶液和过滤该溶液到Falcon管中。 </li><li>使活细胞沉降到试管底部（活细胞沉淀应该采取2-4分钟）。 </li><li>每四个钙溶液（ 表3）到4个独立15毫升Falcon管中的等分试样2.5毫升。 </li><li>再次使用标准的巴斯德移液管小心地从管底除去细胞沉淀，将细胞转移到所述第一对钙的解决方案。 </li><li>使细胞沉降到试管底部并在随后的钙溶液重复步骤1.17，直至细胞是在最后的解决方案。 </li><li>不要让细胞沉淀在4吨ħ钙溶液。盖上管，并把它在其一侧。 </li></ol> 2。细胞培养<ol><li>之前的隔离，板1微克/毫升的天然小鼠层粘连蛋白上的玻璃盖玻片上，并孵育至少1小时，在37℃和2％CO 2下。还可以允许电镀和培养基（ 表4）在孵化器升温，在37℃和2％CO 2下。这允许在媒体中的溶解的CO 2来平衡。 <ol><li>不要从媒体卸下顶部。只需松开顶部，以避免污染。 </li></ol></li><li>让所述分离的细胞沉淀在Falcon试管的底部。 </li><li>使用一个标准的塑料巴斯德吸管，重悬在电镀介质的适量除去沉淀。 </li><li>镀上的层粘连蛋白包被的盖玻片上的细胞在适当的碟子和孵育在37℃和2％CO 2的至少30-60分钟，以使细胞附着在盖玻片上。 </li><li> *如果用腺病毒转染，稀释该病毒在镀介质的适量和替换在与含病毒的培养基的培养皿镀介质。让病毒孵育的细胞2小时，在37℃和2％CO 2。** </li><li>从盘中取出镀介质并更换培养基。 </li><li>每天认真改变培养基，以免扰乱在盖玻片的细胞。 </li></ol>使用这种方法，细胞已被成功地培养长达72小时。培养和GFP转染的细胞的图像可以在图5中找到。 3。 MMSYS系统<ol><li>电源MMSYS系统确保弧灯首先启动。 </li><li>管连接从泵到MMSYS室的适当的入口和出口（ 图6）。 </li><li>素系统与钙缓冲液（B）（ 表2）。 </li><li>放置APPRopriately大小的玻璃盖玻片放入室内，并拧紧（ 图6E）。大多数MMSYS室使用任何22毫米或25平方毫米盖玻片。请咨询您的MMSYS用户手册，以确定适当的盖玻片为您室。 </li><li>转移500μl的细胞溶液到一个小离心管中。 </li><li>加入0.5微升Fura2-AM（1微克/微升原液）的小管细胞，并让他们在黑暗中在室温下孵育5-7分钟。这允许Fura2-AM对通过细胞膜通过快速被动扩散的“负载”到细胞中。 <ol><li>让Fura-2的加载细胞沉淀在管的底部，并且用500μl钙缓冲液B的洗涤过量的Fura </li></ol></li><li>关掉房间的灯。 </li><li>使用标准的巴斯德移液管，滴1-2滴的“装”的细胞溶液进入腔室（ 图6C）的中心（取决于细胞浓度），并允许细胞吨Ø解决到盖玻片5分钟。 <ol><li>将细胞在所述腔室的密度应该是这样的单一的非重叠的小区所用的MMSYS数据采集平台很容易地观看。 </li></ol></li><li>小心地开始通过腔室流到钙缓冲液（B）。 </li><li>提高室内温度37°C。 <ol><li>重要提示：不要打开加热装置，直到流已经启动。这可能会严重损坏反馈thermocoupler（ 图6A）。 </li></ol></li><li>确保该室被连接到MyoPacer通过连接线（ 图6B），开始踱步细胞5 Hz在1.5倍的细胞起搏阈值。 </li><li>选择一个跳动在正确的频率单元格，并将其放入取景光圈。 </li><li>调节摄像机和成帧光圈尺寸，使整个小区在该窗口的中心，朝向水平方向，与肌节AP父。 <ol><li>用于细胞长度调整红色和绿色（右和左）光电二极管指标，该小区的边缘。调整对比度以优化黑/白对比度在小区的边缘。请参阅Ionoptix手册，了解有关详情。 </li></ol></li><li>将盒中包含明确定义的肌节细胞的面积，调整焦距，以优化功率谱（红色）的峰值。也调整显微镜的亮度，以优化蓝色平滑窗口和黑对比度信息。 </li><li>调整绿色门槛条捕捉尽可能多的红色的功率谱（峰值）和一些背景尽可能。 </li><li>开始录制。 </li><li>经过足够的数据已被记录，按一下“暂停”，暂时停止记录，并确保该观察窗的既不是焦点也不尺寸被改变，移动显微镜的阶段到盖玻片，其中没有细胞或细胞物质可以是一个区域见过。莱恩根据研究者的实验方案记录的第g而变化。 <ol><li>注意：点击“停止”，将终止该记录和无背景将能够被记录为单元。 </li></ol></li><li>点击“恢复”录制背景测量。 </li><li>经过足够的背景已被记录点击“停止”，以终止录制。 </li><li>点击“文件&gt;&gt;保存”，保存所有的痕迹该单元在一个单一的，ZPT文件。 </li><li>重复步骤3.12 - 3.20到足够的细胞已被记录。 </li><li>咨询IonOptix-MMSYS用户手册12对记录的数据进行分析说明。野生型心肌特性所记录的数据在图7中示出。 </li></ol> 4。膜片钳不同细胞类型的膜片钳已经很好这轴颈25-28如前所述。因此，我们专注于一些批评家人参数为成人的心肌细胞成功膜片钳用我们的协议中所述隔离。 <ol><li>用移液管牵拉和硼硅酸盐玻璃（长丝：外径1.5mm时，ID0.86 ​​毫米- 10cm长）拉移液管表现出〜2.0-3.0MΩ时充满移液管溶液（ 表5）。 </li><li>火抛光轻轻用成茂或其他垂直火抛光枪头。补丁吸管的电阻应为火抛后2-4个月。 </li><li>打开计算机，放大器（Axopatch 200B），AD转换器（Digidata 1440A，Axon仪器）和微操作机器人（MP-285）上。对于全细胞膜片钳，我们开始与如前面所述标准参数。 </li><li>对培养的成年的CM，从孵化器中移除它们，并轻轻冲洗其上的CM是在室温下镀用PBS盖玻片。 </li><li>轻轻取出盖玻片，并将其放置在灌注室的倒置显微镜（Nikon TE 2000）。 <ol><li>保证进用于灌注系统和出口（用于吸入），只是盖玻片的表面上。 </li></ol></li><li>开始用适当的细胞外液（ 表5）灌注。 </li><li>对于新鲜分离成年的CM，将胶原蛋白包被的盖玻片在灌注系统上面。 <ol><li>因为细胞会在溶液中，轻轻地用胞缓冲液更换溶液。 </li></ol></li><li>使用修改后的巴斯德吸管（ 图4），轻轻悬浮细胞在细胞外的缓冲区，取等分，将它放在盖玻片。 </li><li>让细胞在开始灌注在4.4之前附上10-15分钟。 </li><li>回填用微滤（世界精密仪器，28 G）针与细胞内缓冲的全细胞膜片吸管。确保没有气泡在吸管尖，轻轻拍打，使气泡浮到surfaCE的解决方案。 </li><li>附加填充补丁吸管的吸液管保持器，以确保有微电极和内部溶液之间的接触。 <ol><li>我们使用的氯化银电极，但通过在家用漂白剂中浸渍过夜，取&#39;chloriding&#39;的护理一周的银线至少一次。 </li></ol></li><li>轻轻放下吸管进入浴缸，确保浴缸电极浸没在任何时候洗澡/细胞外液中。抵消任何液体接界电势在这个时候。大路口的潜力可能下降氯化银电极上的标志。 </li><li>健康成人的圆筒状的CM被确定在显微镜和集中在视野中。如先前所述，移动显微操作和调整焦点的组合允许补丁吸管的接近和CM的表面上。 <ol><li>一旦它们在同一焦平面上，我们使用的视觉组合化细胞和测试脉冲（在Axopatch 200B有）。 </li><li>一旦移液管的阻力减小一半（这表明吸管与所述细胞的表面相接触），且细胞继续寻找圆柱形，我们建立使用轻柔抽吸千兆欧姆密封。 </li><li>对于CM的，我们更喜欢用嘴吸建立负压。如果成功获得了gigaseal，我们再次使用温和的节奏用嘴吸，以“磨合”到细胞，建立全细胞膜片模式。 </li></ol></li><li>健康CM的典型静息电位都在-85到-90毫伏的顺序。一旦获得gigaseal，使用电流钳与I = 0 pA的测量静息膜电位。 <ol><li>如果静息膜电位去极化，这代表一个损坏单元或密封不良。 </li></ol></li><li>由于CMS是大细胞有很大的表面积，仔细注意必须支付给电容补偿，尤其是瓦特母鸡快速激活的电流，如钠通道需要进一步研究。如果有足够的电容补偿不能使用获得的内置的脉冲发生器补偿设定，prepulses在亚阈值电压和相反极性可能要被应用和所得到的电流从所记录的信号中减去。这样的协议是很容易编程Axopatch。 </li><li>一旦全细胞膜片模式已经建立，等待15分钟，使达到平衡，并保证密封的稳定性之前开始电压或电流钳协议。我们使用了在这以前已经描述的标准协议和其他期刊和后不会加以阐述。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在这份报告中，我们已经描述了成功分离和成人CM的文化从小鼠心脏所必需的技术。我们的技术可用于使用上述方法CM功能和兴奋性的后续研究。为研究成年CM的功能的关键参数是分离的CM的卫生和质量。如上所述，我们的技术允许以高收率的功能性细胞，适合于操纵用的腺病毒/慢病毒感染的培养和细胞兴奋性和收缩功能的分析基因表达。 一种用于功能成年小鼠CM的隔离的最重...

心血管疾病的小鼠模型曾担任有效的工具，基本阐明疾病机理1,2以及识别潜在的治疗靶点1,3。特别是，利用获取的心脏疾病的两个小鼠模型（如压力负荷）4,5和转基因小鼠模型的具有先进我们的心脏疾病6-8的理解。利用细胞培养技术，研究信号级联反应3,9,10和变更中，在单细胞水平的背后细胞的兴奋性和兴奋-收缩偶联在心脏11-13个别蛋白质已补充了体内小鼠模型。然而，由于缺乏反映成人CM的结构和功能足够的细胞系已经显著限制。研究者已经寻求通过研究个体的蛋白质，如离子通道，以克服这一点，在异源表达系统<suP&gt; 14，虽然这些研究为我们提供了有用的信息，在离子通道生物物理学或蛋白质运输，CM的原生微环境的代表性不足而言是一个显著的限制。其次，因为大多数这些异源细胞不具有成熟的收缩装置，但一直未能学习的收缩功能和细胞兴奋性和收缩之间的复杂的相互作用。为此，研究人员已经转向原发性心脏细胞培养的许多它们的体外功能研究的。最后，分离的心肌细胞研究允许收缩功能的评估，而无须多细胞制备的混杂因素包括疤痕或纤维化和纤维取向的影响。 小学新生大鼠心室肌细胞（NRVMs）是比较容易培养，可以感染腺病毒和慢病毒操纵基因的表达15，第二因此，已经成功1使用，但有自己的限制。虽然他们提供了一个生理微环境1，并一直信令领域的主力，形态和NRVMs和成人cardiomyoctyes的亚细胞组织间的巨大差异使他们对离子通量和兴奋-收缩耦联在成人心脏调查不足的模型。最值得注意的是NRVMs缺乏一个明确的T管状子系统4。由于钙离子通量和动态都严重依赖于成熟的T管和肌浆网（SR）结构6，Ca 2 +的动力学和NRVMs心肌收缩功能的研究是不是在成人的心肌这些关键流程的准确反映。另外，信号通路的一些部件新生和成年小鼠9之间的不同，从而提供了另一个限制用于 ​​研究疾病过程和其我MPACT在NRVMs细胞的兴奋性和收缩性。最后，该收缩机构的分布导致多方向和不均匀的细胞缩短限制收缩测量的精度。 因此，使用分离的成年心肌细胞提供了更准确的体外模型系统。知识的超常增长成为可能，小鼠遗传操作得到强调功能的心肌细胞分离小鼠的意义。事实上，成人的CM从小鼠模型中分离的特性也已摆脱了许多生物和病理事件的光。从转基因小鼠模型中分离的CM已经允许用于在疾病模型如缺血/再灌注17,18上的单个细胞2,16的收缩特性的增益或蛋白质的功能丧失，和生存能力的研究，从而补充从获得的信息体内研究对SE小鼠。从天性心脏疾病3,19,20小鼠模型（如横向主动脉缩窄致压力过载，模仿高血压或主动脉瓣狭窄）或运动5,21（用于模拟生理性肥大）允许检查使用隔离成年的CM的牵连在这些过程与在单细胞水平的细胞的兴奋性和兴奋 - 收缩偶联信号传导级联的相互作用。此外，使用腺病毒驱动的基因表达在成人的CM操纵基因表达的能力，使我们有机会来剖析复杂的信号通路的组成部分。 从电的角度来看，整个电池的电压和上分离的成CM的电流钳实验中阐明的离子通量中的基线的性质和各种疾病状态是至关重要的。由于细胞膜的结构复杂和差动保护制服的在成人的CM和NRVMs或异源的细胞系之间的脚手架结构，修补成年细胞的能力给出了某些膜蛋白，结构蛋白，并在成年心脏的电组件的离子通道相互作用伙伴的效果好得多表示。 尽管在研究成年鼠心肌细胞，分离培养成年小鼠心肌细胞已经被挑战，敦促需要的方法来隔离小鼠存活心肌，并保持他们的文化，以允许进一步的遗传操作使用病毒的系统，准确地描述这种突出的优点向量。以前的研究已经使用，也可以急性分离的小鼠成年CMS或培养的大鼠成年CMS。后者更容易培养比成年鼠的CM，和大多数实验在体外操纵基因的表达已经用大鼠成年CMS。一些研究已经成功地改变和调查FUNCTI在成年小鼠的CM ONAL基因表达，在实验的范围呈现一个大的限制。因此，在这里我们将详细介绍这些方法，从以前的调查修改，为隔离7,8,22，文化3,10,15,23，腺病毒感染11-13,15和成年小鼠脑室cardiomyoctyes功能分析。这种隔离协议导致Ca 2 +的耐性，我们已经成功地培养长达72小时，瞬时转染腺病毒兴奋心肌细胞。这些分离的细胞的功能性可使用MMSYS成像系统14,24和膜片钳，这也将讨论进行评估。

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Phosphate 			Monobasic</td>
 <td>Sigma</td>
 <td>S8282</td>
 <td></td>
 </tr>
 <tr>
 <td>D-glucose minimum</td>
 <td>Sigma</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>Sigma</td>
 <td>T0625</td>
 <td></td>
 </tr>
 <tr>
 <td>2,3-Butanedione 			monoxime</td>
 <td>Sigma</td>
 <td>B0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B</td>
 <td>Roche Applied 			Science</td>
 <td>11088807001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche Applied 			Science</td>
 <td>11088858001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Protease 			XIV from Streptomyces griseus</td>
 <td>Sigma</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from Bovine 			Serum</td>
 <td>Sigma</td>
 <td>A2153</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Sigma</td>
 <td>C8106</td>
 <td></td>
 </tr>
 <tr>
 <td>Minimum Essential 			Media</td>
 <td>Sigma</td>
 <td>51411C</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin solution 			from bovine serum</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Sigma</td>
 <td>G3126</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Sigma</td>
 <td>P4333</td>
 <td></td>
 </tr>
 <tr>
 <td>Insulin-transferrin-sodium 			selenite media supplement</td>
 <td>Sigma</td>
 <td>I1884</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Chloride</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sial/289329?lang=en&amp;region=US" target="_blank">289329</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Sigma</td>
 <td>G3291</td>
 <td></td>
 </tr>
 <tr>
 <td>Adenosine 			5'-triphosphate magnesium salt</td>
 <td>Sigma</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene 			glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 			acid</td>
 <td>Sigma</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Hydroxide 			Solution</td>
 <td>Sigma</td>
 <td>232041</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetraethylammonium 			hydroxide solution</td>
 <td>Sigma</td>
 <td>86643</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiVisor optical 			glass binocular 			visor</td>
 <td>Dohegan Optical 			Company Inc.</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue 			forceps, 5.5", 1x2 teeth</td>
 <td>Roboz Scientific</td>
 <td>RS-8164</td>
 <td></td>
 </tr>
 <tr>
 <td>Moloney 			forceps - 4.5" (11.5 cm) long slight curve, serrated </td>
 <td>Roboz Scientific</td>
 <td>RS-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont 			#3 Forceps, Dumostar, tip size 0.17 x 0.10mm</td>
 <td>Roboz Scientific</td>
 <td>RS-4966</td>
 <td></td>
 </tr>
 <tr>
 <td>Packer 			Mosquito Forceps 5" Straight Flat</td>
 <td>Roboz Scientific</td>
 <td>RS-7114</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro 			Dissecting Scissors 4.5" Curved Sharp/Sharp </td>
 <td>Roboz Scientific</td>
 <td>RS-5917</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm</td>
 <td>Roboz Scientific</td>
 <td>RS-5907</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

在培养中使用的主要的心肌细胞（CMS）中提供了一个强有力的补充，以心脏疾病的小鼠模型在推进我们的心脏疾病的认识。特别是，研究离子平衡，离子通道的功能，细胞的兴奋性和兴奋 - 收缩耦联及其改建患病状况及致病突变的能力，导致显著见解心脏疾病。此外，由于缺乏足够的永生化细胞系，以模仿成人的CM，并且新生儿的CM的限制（其缺乏许多成人CM的结构和功能的生物力学特性的）中培养，阻碍了我们的信号转导途径之间的复杂的相互作用的理解，离子通道和在成人心脏加强学习成人的心肌细胞分离的重要性收缩特性。在这里，我们提出了分离，培养，操纵基因表达的腺病毒EXPRE方法ssed蛋白，并从成年小鼠心肌细胞其后的功能分析。使用这些技术将有助于开发机械洞察信号通路调节细胞的兴奋性，Ca 2 +的动力学和收缩，并提供一个更相关的生理表征心血管疾病。

成人cardiomyoctyes结果在棒状，横纹肌和静态（不自发地跳动）的细胞（ 图5A）的隔离。死细胞会显得圆润，无条纹将出席。静止细胞可以培养和转染腺病毒操纵基因的表达（ 图5B和5C）。在培养24小时，活细胞的形态不改变，他们仍然是Ca 2 +的宽容，他们可以通过现场的刺激来节奏。与提出的方法，80-90％的活细胞的产率可以达到。 <p class="jove_content...

Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

在这里，我们描述了使用Langendorff灌流系统成年小鼠cardiomyoctyes隔离。所得到的细胞是钙耐受性，电静态和可培养和转染腺病毒或慢病毒操纵基因的表达。它们的功能也可以使用MMSYS系统和膜片钳技术进行分析。

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-culture-functional-characterization-adult-mouse

Research

JoVE Journal

Biology

24.2K 次观看.  Beth Israel Deaconess Medical Center, Harvard Medical School. 在这里，我们描述了使用Langendorff灌流系统成年小鼠cardiomyoctyes隔离。所得到的细胞是钙耐受性，电静态和可培养和转染腺病毒或慢病毒操纵基因的表达。它们的功能也可以使用MMSYS系统和膜片钳技术进行分析。 

视频: Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

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 ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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 ...

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 ...

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 and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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 ...