We thank Dr. Christian Rickert for critical reading of the manuscript. This work was supported by a grant from the National Heart Lung and Blood Institute (R01-HL088427) to CP. EJS was supported by 5T32-AG000279 from the National Institute on Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

所有的动物程序符合由美国科罗拉多州安舒茨医学校区的大学机构动物护理和使用委员会批准的方案进行。标准协议下一直使用雄性C57BL / 6J小鼠的2-3个月的年龄进行了优化。 1.准备解决库存和用品在实验中的研究进展注:请参阅材料表进行必要的设备和用品。 <ol><li>全台氏，低钙 /镁离子台氏，卡夫改良，Brühe（KB）解决方案，与牛血清白蛋白（BSA）解决方案:如表1所示，准备每个以下方法解决1升。使用过滤超纯去离子水中所有的解决方案。划分为长达六个月各溶液到50ml等分试样并储存在20℃。立即实验前解冻个人等份，并存储于4最多一周6℃。 </li><li>在超纯过滤的去离子水（ 见表1）溶解氯化钠和氯化钙制备50毫升的10mM NaCl和1.8毫米氯化钙2适应解的。在室温下储存长达六个月。 </li><li>吹打入微量离心管中制备弹性蛋白酶4.75酶活性单位（U）等分试样。储存在4℃下长达三个月。 </li><li>吹打成离心管准备375微克等分（超纯H 2 O）胶原酶蛋白酶荟萃。储存在-20°C至三个月。 </li><li>准备两个火抛光的巴斯德吸液管，一个用于组织转移（〜1.5毫米最终开口直径; 图1Aiv和图1C）和一个用于解离（〜直径2毫米; 图1Aiii和图1C）。 <ol><li>得分巴斯德移液器用玻璃切割器和沿刻痕对齐，以产生大于所需尺寸稍大的开口。火- 波兰语各吸管的切断端为〜30-60秒以上上本生灯以产生厚的抛光壁和所需直径的开口的低火焰。确保火抛光开无任何裂缝或粗糙的边缘。 </li></ol></li><li>通过加入准备两个夹层菜约25mL硅氧烷弹性体，根据制造商的指示对各100毫米的培养皿（ 图1AI和图1B）混合。允许在室温下固化48小时。 </li><li>文化只有:25-50准备每毫升电镀液和培养液按表2商店为最多两个星期在4℃。 </li></ol> 2.准备解决方案中使用的细胞分离日注:以下金额从一个鼠标窦房结细胞的分离。 <ol><li>加入2.5倍mL的低的Ca 2+ / Mg 2+离子的台罗德氏（pH为6.9），以每三个小，圆底培养管的秒。地方管在35±1℃水浴。 </li><li>加入2.5倍mL的低钙 /镁离子台氏到一个大圆底文化管。对此管中，加入1等份（4.75 U）弹性蛋白酶和一个等份（375微克）胶原酶蛋白酶结合。涡流混合。在35±1℃的水浴地方管。 </li><li>添加2.5毫升KB培养基至三个附加的小，圆底培养管和一个附加大圆底培养管中。地方管在35±1℃水浴。 </li><li> 〜7个毫升BSA溶液加入到一个大底的培养管。保持此管在室温下。 </li><li>放置20-40毫升完成台氏溶液的在50毫升的烧杯中（ 图1Aii）。添加10 USP / ml肝素，漩涡混合，然后将烧杯在35±1℃水浴。 </li></ol> 3.准备其他解决方案和材料培养细胞（跳过这些步骤为急性分离的细胞） <ol><li>在无菌组织培养罩，将每只小鼠2个12毫米的圆形玻璃盖玻片上成24孔板的各个孔中。 注:24孔板被使用，因为孔是一个方便的大小，它用于限制用于随后的病毒感染的体积。 </li><li>吸管大约200μl的小鼠层粘连蛋白的100纳克/毫升的溶液在无菌磷酸盐缓冲盐水（PBS）稀释到每个盖玻片。 </li><li>孵育层粘连蛋白盖玻片在培养箱隔离（至少1小时）的在37℃下的持续时间。 </li><li>预暖的电镀培养基和培养基（来自步骤1.7和表3）至37℃。 </li></ol> 4.窦房结隔离<ol><li>在化学通风橱中，放置一个鼠标在两腔室箱的一个室并通过棉签引入另一个室〜200μl的液体异氟烷麻醉。确认麻醉（通常在30〜60〜秒）用脚趾捏。安乐死通过颈脱位鼠标。 注:空1毫升枪头盒可用于形成两腔盒;打开机架倒挂在框中创建单独的隔间为异氟醚，以防止老鼠直接接触它。 </li><li>胸部用剪刀样肋骨取出皮草揭露使用外部组织钳（ 图1Aviii）和解剖剪刀（ 图1Avix）胸腔。洗澡胸腔用约2ml温热完成台氏使用肝素移液管。 注:继续洗澡胸腔必要的时候，不要让准备干。 </li><li>在解剖显微镜下，小心地取出肺和胸腺内部使用剪刀（ 图1Avi）和解剖钳（ 图1Avii）。 </li><li>虽然轻轻地捧着与内部解剖钳心脏的顶点，小心切开下腔静脉和一奥尔塔与内部剪刀从胸腔取出心脏。的心脏转移到的硅解剖的菜肴之一，并与洗澡〜4毫升回暖肝素完整台氏用移液管。 </li><li>东方的心脏，使血管后可见并朝上，与实验者的右动物的右心房和左心房对实验者的左边。一旦为导向，通过顶点到硅解剖盘固定钉的心脏。 </li><li>找到心室和心房（心室上面清晰圈）之间的凹槽。 </li><li>使用内部解剖剪，使一个切口在槽，保持更靠近心室比心房。冲洗槽与切口额外的温暖肝素完整台氏根据需要使心房和阀门的一个明确的说法。继续沿着槽切成心房从心室中分离出来。 </li><li>转移心房组织到第二硅解剖盘和洗澡用〜3毫升回暖肝素完成台氏。 </li><li>定向的组织，使得动物的右心房，现可实验者的左侧，左心房是在右侧。 注:右心房是比较透明的，而左心房有更多的暗红色基调。 </li><li>通过下和优越的腔静脉与右和左心耳针组织，轻轻拉伸的制备。删除任何剩余的脂肪或其他组织，让准备（注意不要切进心房壁，由于窦房结是相当微妙的，可以很容易损坏）的一个明确的说法。 </li><li>通过经由腔静脉切开心房前壁。重新定位销必要形象化房间隔。 </li><li>沿房间隔切割以除去左心房。重新钉住准备，轻轻地伸展。 </li><li>除去叔他右心耳并通过沿嵴终，这显示为深橙色条纹接壤心耳切割释放窦房结。 </li><li>再针的淋巴组织和削减它横向（垂直于嵴），以产生三个相等大小的条带。 </li></ol> 5.窦房结消化<ol><li>使用窄火抛光的吸管（ 图1Aiv），窦房结组织的三个条带转移到第一含2.5低的Ca毫升三小，圆底管2+ / Mg 2+离子蒂罗德在35±1°水浴。孵育5分钟。 </li><li>转移组织带以第二小圆底含有2.5 mL的低的Ca管2+ / Mg 2+离子蒂罗德在35±1℃的水浴中，使用相同的窄吸管。通过轻轻旋转管或通过狭窄的吸管轻轻吹打洗净组织带。不要INVERt为管。 </li><li>转移组织带向第三小圆底含2.5 mL的低的Ca 2+管/镁离子台氏，并重复步骤5.2中所述的清洗步骤。 </li><li>转印条带成含有2.5低的Ca毫升大圆底管2+ / Mg 2+离子蒂罗德与35℃水浴中的酶（蛋白酶加胶原酶蛋白酶掺合物）。确保所有这三个组织带都存在。孵育在35±1℃下10-15分钟。通过轻轻摇动混合管每5分钟。不要颠倒管。 </li></ol> 6.窦房结肌细胞解离<ol><li>以下的酶消化，使用窄火抛光移液器在35±1℃下的组织带轻轻转移到含2.5毫升的KB溶液中的第一小，圆底管中。通过轻轻摇动管冲洗组织。 注:组织带将出现一定程度的半透明并可能在日聚集在一起，是点。把手轻轻酶消化后，以避免失去细胞。 </li><li>在35±1℃的转移组织含有2.5毫升KB第二小圆底管中。摇动轻轻地洗。 </li><li>在35±1℃的转移组织含有2.5毫升KB第三小圆底管中。摇动轻轻地洗。 </li><li>在35±1°C转移组织含2.5毫升KB大，圆底管。 </li><li>使用较大的火抛光的吸管（ 图1Aiii和图1C），游离由恒研磨大圆底管细胞在约0.5-1赫兹5-10分钟，注意保持淹没解离管35±1℃的水浴中，并避免引入气泡到溶液中。 注意:研磨时间与离解吸管直径和移液的力而变化。时间应调整，以使组织块在t剩余他解离年底显得单薄，透明，纤细。如果组织保留任何颜色，解离可能是不完整的。频率（0.5-1赫兹）是用手工确定。 </li><li>除去从水浴含有解离的SAM圆底管中，在室温下平衡5分钟。 </li></ol> 7.窦房结钙重新适配（在室温下进行） 注:对于自组装往培养实验，在下面的部分中的过程应该在无菌组织培养罩来进行。如果自组装将被用于急性实验，没有必要在无菌环境中执行这些步骤。 <ol><li>添加75微升的氯化钠/ 氯化钙适配溶液（ 表2）。摇动轻轻地混合和孵育5分钟。 </li><li>加入160微升氯化钠/ 氯化钙适应的解决方案。摇动轻轻地混合和孵育5分钟。 </li><li>加入390微升BSA溶胶ution（ 见表2）。摇动轻轻地混合和孵育4分钟。 </li><li>加将1.25ml BSA溶液。摇动轻轻地混合和孵育4分钟。 </li><li>加入4.37毫升的BSA溶液。摇动轻轻地混合和孵育4分钟。 注意:钙的终浓度将是1.8毫米。 </li><li>以下钙重新适应，通过允许在〜2000×g离心〜10分钟或通过离心进行重力沉降3分钟收集自组装膜。 <ol><li>对于急性分离细胞，温和地去除和丢弃约5ml用玻璃巴斯德吸管取上清液，留下细胞的〜2毫升的体积。存储这些细胞在室温下长达〜8小时，对膜片钳记录。 </li><li>对于培养的细胞，用无菌玻璃巴斯德吸管除去尽可能多的上清液越好。在1ml悬浮细胞沉淀预热（37℃）镀层中（ 表2）。 </li></ol></li></ol> 8.电镀和文化窦房缪的核细胞（跳过了急性分离的细胞） <ol><li>来自步骤3.3用巴氏吸管去除盖玻片层粘连蛋白溶液。立即种子500微升（〜50-100个细胞）到每个层粘连蛋白涂层的盖玻片（来自步骤3）。 注:收缩抑制剂2,3-丁二酮单肟（BDM）被包括在电镀和培养基，以防止收缩，这将导致细胞9的磨损。 </li><li>返回包含新接种的SAM的24孔板的孵化并在95％空气/ 5％CO 2的气氛中，在37℃保持。允许细胞粘附于盖玻片在电镀介质4-6小时（ 表2）。 </li><li>轻轻地用无菌巴斯德吸管去除电镀液。每个预温（37℃）培养基的井500微升（ 表2）替换。 </li></ol> 9.成人窦房肌细胞文化的腺病毒转导（跳过了急性分离的细胞） <ol><li>估计CE数每盖玻片LLS立即通过计数细胞中的视场，在显微镜下，调整放大率施加腺病毒之前。计算所有细胞，而不仅仅是地对空导弹。 </li><li>稀腺病毒在培养基199等1-10微升的该应用程序需要达到的感染的每个细胞100病毒单位的最终复数（MOI）调节稀释的病毒滴度。添加以逐滴方式腺病毒溶液直接在镀自组装膜。 </li><li>孵育与含病毒的培养基过夜（〜12-14小时）的细胞。然后用新鲜培养基交换。维持细胞在培养箱，改变培养基每48小时，直到获得期望的蛋白质表达。 </li></ol> 10.急性分离或养殖地对空导弹的功能评价注:以下协议是用两性霉素穿孔，补丁技术，同时记录自发的AP和我孤立地对空导弹的功能性评估的一个例子<sub&gt; f起相同的细胞（见参考文献9）。 <ol><li>如表3中所述制备的记录方案。 </li><li>制备20毫克/毫升两性霉素-B的DMSO上记录的当日新鲜的储备溶液。保持库存在室温和避光。稀两性霉素-B库存胞内溶液至200微克的终浓度/ ml的临使用前。保持冰最终吸管解决方案，并避光。 注:对于实验的含两性霉素吸管溶液应准备通过稀释储备溶液的等分进入胞内溶液和涡流至少1分钟新鲜每小时。 </li><li>转移的等分试样急性分离的SAM细胞悬液或玻璃盖玻片在35±1℃的轴承培养的SAM至含有台氏液的记录室的片段。灌注细胞用台氏液至少2分钟之前，电recordinGS去除培养基中剩余的任何残留BDM。 注:自发收缩应当在转移到台氏液立即明显。自组装用于记录可以通过功能，包括自发收缩活性，形态特征（ 例如 ， 图2），缺乏条纹的，HCN4蛋白的表达，I F电流的存在，膜电容&lt;50 pF时，自发的AP的组合来确定与波形，包括一个舒张去极化阶段和缓慢的冲程。 </li><li>用硼硅玻璃移液器1.5-3.0MΩ的电阻，与细胞内液填充尖端而缺少浸渍10-30秒霉素。然后再填充与含两性霉素溶液吸管。 GΩ的细胞贴附密封应尽可能快得越好。如果密封的形成是困难的，增加的顶端填充时间。 注:访问电阻应不断监视下形成细胞贴附密封的，和录音应该只在&lt;10MΩ稳定的接入电阻后展开。 </li><li>要记录自发的AP，开关放大器快速电流钳模式，无电流注入。 注意:记录的AP时，以稳定的燃烧率，如先前报道10为1nM异丙被包括在细胞外台氏液。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapidly+activating+delayed+rectifier+K++current+regulates+pacemaker+activity+in+adult+mouse+sinoatrial+node+cells.">A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells.</a> Am J Physiol Heart Circ Physiol. 286 (5), H1757-H1766 (2004).</li><li>Mangoni, M., Nargeot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+hyperpolarization-activated+current+(I(f))+in+isolated+mouse+sino-atrial+cells.">Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells.</a> Cardiovasc Res. 52 (1), 51-64 (2001).</li><li>Cho, H. S., Takano, M., Noma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrophysiological+properties+of+spontaneously+beating+pacemaker+cells+isolated+from+mouse+sinoatrial+node.">The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node.</a> J Physiol. 550 (Pt 1), 169-180 (2003).</li><li>Rose, R. A., Lomax, A. E., Kondo, C. S., Anand-Srivastava, M. B., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+C-type+natriuretic+peptide+on+ionic+currents+in+mouse+sinoatrial+node:+a+role+for+the+NPR-C+receptor.">Effects of C-type natriuretic peptide on ionic currents in mouse sinoatrial node: a role for the NPR-C receptor.</a> Am J Physiol Heart Circ Physiol. 286 (5), H1970-H1977 (2004).</li><li>Rose, R. A., Kabir, M. G., Backx, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Heart+Rate+and+Sinoatrial+Node+Function+in+Mice+Lacking+the+cAMP+Regulator+Phosphoinositide+3-Kinase-\gamma\.">Altered Heart Rate and Sinoatrial Node Function in Mice Lacking the cAMP Regulator Phosphoinositide 3-Kinase-\gamma\.</a> Circ Res. 101 (12), 1274-1282 (2007).</li><li>Hua, R., Adamczyk, A., Robbins, C., Ray, G., Rose, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+constitutive+phosphodiesterase+activity+in+mouse+sinoatrial+node+and+atrial+myocardium.">Distinct patterns of constitutive phosphodiesterase activity in mouse sinoatrial node and atrial myocardium.</a> PloS ONE. 7 (10), e47652 (2012).</li><li>Groenke, S., Larson, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+atrial-specific+knockout+of+sodium-calcium+exchange+eliminates+sinoatrial+node+pacemaker+activity.">Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.</a> PloS ONE. 8 (11), e81633 (2013).</li><li>Torrente, A. G., Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burst+pacemaker+activity+of+the+sinoatrial+node+in+sodium-calcium+exchanger+knockout+mice.">Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice.</a> Proc Nat Acad Sci USA. 112 (31), 9769-9774 (2015).</li><li>Denyer, J. C., Brown, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+sino-atrial+node+cells:+isolation+and+electrophysiological+properties.">Rabbit sino-atrial node cells: isolation and electrophysiological properties.</a> J Physiol. 428 (1), 405-424 (1990).</li><li>Thum, T., Borlak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butanedione+monoxime+increases+the+viability+and+yield+of+adult+cardiomyocytes+in+primary+cultures.">Butanedione monoxime increases the viability and yield of adult cardiomyocytes in primary cultures.</a> Cardiovasc Toxicol. 1 (1), 61-72 (2001).</li><li>Borlak, J., Zwadlo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myosin+ATPase+inhibitor+2,3-butanedione+monoxime+dictates+transcriptional+activation+of+ion+channels+and+Ca(2+)-handling+proteins.">The myosin ATPase inhibitor 2,3-butanedione monoxime dictates transcriptional activation of ion channels and Ca(2+)-handling proteins.</a> Molec Pharmacol. 66 (3), 708-717 (2004).</li></ol>

本文提出了从成年小鼠完全分化的窦房结细胞的分离和培养的详细协议。隔离协议可靠生产适合要么直接电生理分析或随后的培养自发活动的鼠标地对空导弹。类似的协议已经报道许多其它基团（例如，参见参考文献11,12,10,13-17）。然而，我们的用于体外维持成年小鼠的SAM协议保留特性形态，自发活动，细胞的电生理特性，并允许蛋白9的腺病毒递送。 ?...

在心脏的窦房结起搏细胞（窦房结细胞"地对空导弹"）产生自发的，有节奏的动作电位（APS），通过心肌传播发起每次心跳。使用急性分离地对空导弹许多种实验已经为有助于心脏起搏器活动的产生机制，阐明至关重要。地空导弹是从他们的心房和心室肌同行中的形态，功能和表达方面有很大不同高度专业化的心肌细胞。在自组装膜自发的AP的标志是舒张驱动的膜电位向阈值，以触发下一个AP 1,2-期间自发去极化。这种"起搏器势"取决于许多不同的膜电流包括"滑稽电流"（我f）中 ，T型和L型钙电流，钠钙交换CURR的协调活动耳鼻喉科（ⅠNCX），这是由钙释放从肌质网3,4驱动。 虽然急性分离的小鼠地对空导弹是起搏的研究必不可少的实验准备，地空导弹的小鼠隔离可以通过一个具有挑战性的方法，因为鼠标SAN的模糊的解剖和小尺寸需要细致入微的显微切割和组合的酶和机械细胞解离需要仔细优化。 在这里，我们提供已被成功地用于从膜片钳记录5-8成年小鼠隔离地对空导弹的协议的详细视频演示。据我们所知，没有这样的视觉可从任何其他来源的示范。此外，一个新的方法，证明了在该分离的SAM从成年小鼠可在体外维持数天，由此允许引入蛋白质，基因编码通过腺病毒感染9报告分子或RNAi。

<table><tbody><tr><td>Sylgruard/Elastomer Kit</td><td>Dow Corning</td><td>184 SIL ELAST KIT 0.5KG</td><td></td></tr><tr><td>Borosilicate 9&quot; pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20C</td><td></td></tr><tr><td>Small, round bottomed culture tubes</td><td>Fisher Scientific</td><td>352059</td><td></td></tr><tr><td>Large, round bottomed culture tubes</td><td>Corning</td><td>14-959-11B</td><td></td></tr><tr><td>Elastase</td><td>Worthington Biochemical</td><td>LS002279</td><td></td></tr><tr><td>Liberase TM</td><td>Roche</td><td>5401119001</td><td>Tissue dissociation solution</td></tr><tr><td>Heparin</td><td>SAGENT Pharmaceuticals&amp;nbsp;</td><td>NDC 25021-400-10</td><td></td></tr><tr><td>Mouse Laminin</td><td>Corning</td><td>CB-354232</td><td></td></tr><tr><td>12 mm round glass coverslips</td><td>Fisher&amp;nbsp;</td><td>12-545-80</td><td></td></tr><tr><td>24-well culture plate</td><td>Fisher</td><td>08-772-1</td><td></td></tr><tr><td>Ad-mCherry</td><td>Vector Biolabs</td><td>1767</td><td></td></tr><tr><td>Ad-eGFP</td><td>Vector Biolabs</td><td>1060</td><td></td></tr><tr><td>Plastic, disposable transfer pipette</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Micro scissors</td><td>Fisher Scientific</td><td>17-467-496</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Roboz Instruments</td><td>RS-4904</td><td></td></tr><tr><td>Tissue Forceps</td><td>Roboz Instruments</td><td>RS-8164</td><td></td></tr><tr><td>Dissecting Iris Scissors</td><td>WPI, Inc.</td><td>501264</td><td></td></tr><tr><td>Dissecting Pins</td><td>Fine Science Tools</td><td>26002-20</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>71376</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>60128</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>60353</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>54457</td><td></td></tr><tr><td>glucose</td><td>Sigma</td><td>G0350500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>taurine</td><td>Sigma</td><td>T0625</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>K-glutamate</td><td>Sigma</td><td>G1501</td><td></td></tr><tr><td>K-aspartate</td><td>Sigma</td><td>A6558</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>creatine</td><td>Sigma</td><td>C0780</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Amphotericin-B</td><td>Fisher Scientific</td><td>1397-89-3</td><td></td></tr><tr><td>Isoproterenol</td><td>Calbiochem</td><td>420355</td><td></td></tr><tr><td>Media199</td><td>Sigma</td><td>M4530</td><td></td></tr><tr><td>2,3-butanedione monoxime (BDM)</td><td>Sigma</td><td>B0753</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>SH30071</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A5611</td><td></td></tr><tr><td>Insulin&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>I3146</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>I3146</td><td></td></tr><tr><td>Selenium</td><td>Sigma</td><td>I3146</td><td></td></tr><tr><td>Penicillin</td><td>GE Healthcare</td><td>SV30010</td><td></td></tr><tr><td>Streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr></tbody></table>

methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice

窦房结细胞（SAMS）作为心脏的起搏器自然，倡导通过产生自发的动作电位（APS）击败每一个心脏。这些起搏器的AP反映许多膜电流和细胞内钙循环的协调活动。然而，推动自发起搏器活动地对空导弹的确切机制仍然难以实现。急性分离的地对空导弹是实验解剖心脏起搏的分子基础的必要准备。然而，模糊的解剖，显微解剖复杂和挑剔的酶消化条件阻碍了广泛使用急性分离地对空导弹。此外，方法无法获得直到最近允许地对空导弹的长期培养的蛋白表达研究。在这里，我们提供了地对空导弹从成年小鼠中分离了一步一步的协议和视频演示。方法也证明了体外和expressi维持成年小鼠地对空导弹通过对腺病毒感染外源性蛋白质。急性分离，并通过这些方法制备培养的地对空导弹是适用于各种电生理和影像学检查。

这里所描述的协议已被预先采用自发活性的SAM从成年小鼠适合于各种不同的膜片钳研究5-8的隔离。此外，该协议允许，可以在培养物中维持长达一周孤立的SAM。基因转移到培养的细胞可以通过腺病毒感染9来完成。在本节介绍的结果我们以前的工作中获得，并在这里显示为急性分离培养地对空导弹的特点例子。 ...

Watch this Scientific Journal Video about Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice at JoVE.com

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

方法证实，用于从膜片钳电生理学或成像研究成年小鼠窦房结细胞（自组装膜）的分离。分离的细胞可以直接使用，也可以在培养物中保持，以允许所关注的蛋白质，如遗传编码的记者的表达。

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). ...

methods-for-isolation-culture-functional-characterization-sinoatrial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.1K 次观看.  University of Colorado Anschutz Medical Campus. 方法证实，用于从膜片钳电生理学或成像研究成年小鼠窦房结细胞（自组装膜）的分离。分离的细胞可以直接使用，也可以在培养物中保持，以允许所关注的蛋白质，如遗传编码的记者的表达。 

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

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 ...

科研

生物学

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 ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

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 ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

微电极阵列记录中天节点发射率，以识别小鼠内在心脏起搏缺陷

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

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 ...

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. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

从小鼠中分离心肌细胞的安特格拉德灌注方法

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

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