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. 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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 border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sylgruard/Elastomer Kit</td>
			<td style="width:167px;">Dow Corning</td>
			<td colspan="2" style="width:237px;">184 SIL ELAST KIT 0.5KG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Borosilicate 9&#34; pasteur pipettes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Small, round bottomed culture tubes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">352059</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Large, round bottomed culture tubes</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">14-959-11B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Elastase</td>
			<td style="width:167px;">Worthington Biochemical</td>
			<td style="width:175px;">LS002279</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Liberase TM</td>
			<td style="width:167px;">Roche</td>
			<td style="width:175px;">5401119001</td>
			<td>&#160;Tissue dissociation solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Heparin</td>
			<td style="width:167px;">SAGENT Pharmaceuticals&#160;</td>
			<td style="width:175px;">NDC 25021-400-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Mouse Laminin</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">CB-354232</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">12 mm round glass coverslips</td>
			<td style="width:167px;">Fisher&#160;</td>
			<td style="width:175px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">24-well culture plate</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:175px;">08-772-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-mCherry</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1767</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-eGFP</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1060</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Plastic, disposable transfer pipette</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Micro scissors</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">17-467-496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dumont #4 Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Tissue Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-8164</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Iris Scissors</td>
			<td style="width:167px;">WPI, Inc.</td>
			<td style="width:175px;">501264</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Pins</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:175px;">26002-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma</td>
			<td>60128</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">KH2PO4</td>
			<td>Sigma</td>
			<td>60353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-glutamate</td>
			<td>Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-aspartate</td>
			<td>Sigma</td>
			<td>A6558</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">creatine</td>
			<td>Sigma</td>
			<td>C0780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Amphotericin-B</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">1397-89-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Isoproterenol</td>
			<td style="width:167px;">Calbiochem</td>
			<td style="width:175px;">420355</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Media199</td>
			<td>Sigma</td>
			<td style="width:175px;">M4530</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">2,3-butanedione monoxime (BDM)</td>
			<td>Sigma</td>
			<td style="width:175px;">B0753</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td style="width:175px;">SH30071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td style="width:175px;">A5611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Insulin&#160;&#160;</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Transferrin</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Selenium</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin</td>
			<td>GE Healthcare</td>
			<td style="width:175px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Hyclone</td>
			<td style="width:175px;">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). ...

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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

Primary Culture of Adult Rat Heart Myocytes

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

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

成年大鼠心脏心肌细胞的原代培养

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

科研

生物学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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

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

成人的心肌细胞的分离和遗传操作共聚焦成像

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

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

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

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

分离，培养和成年小鼠Cardiomyoctyes的功能分析

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

Isolation and 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 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 ...