This work was supported by the British Heart Foundation, project grant PG/13/69/30454.

动物工作授权和纽卡斯尔大学伦理委员会的批准。所有动物的程序进行，符合从2010年指令欧洲议会/ 63 / EU的指导原则上用于科学目的的动物的保护。 1.管理的EdU <ol><li>在无菌盐水中的溶液溶解的EdU（0.9％重量/体积）以12.5毫克/毫升的最终浓度。 <ol><li>加热该溶液至40℃，并涡旋以完全溶解。储存足够的教育/盐水在4℃注入所有小鼠在研究允许每天注射6。 注意:通常在6只小鼠都需要每个实验组。然而，功率计算应独立研究完成。 </li></ol></li><li>称量单个小鼠。计算所需的体积施用100微克/克的学科/生理盐水溶液，以每只小鼠（8微升每克库存的EdU溶液）。 </li><li>得出相应的volum教育/生理盐水电子与将25g针头注射器胰岛素。 </li><li>在处理使用经批准的家庭办公室技术小鼠腹腔内执行（ip）注射。 <ol><li>放置在笼盖鼠标。轻轻地向后拉动尾部的基础上，并颈背紧紧抓住鼠标。 </li><li>通过按住鼠标用食指和拇指头的根部附近和向后倾翻动物的头朝向地板暴露于注射腹部。 </li><li>擦拭用70％酒精溶液腹部。找到动物的中线和右下象限。注入学科/盐溶液到动物到右下象限。 </li><li>注射小鼠用EdU /生理盐水溶液，并记录每天的时间。 </li></ol></li><li>在一天的同一时间重复步骤1.2至1.4，连续6天。 注意:作为一个的EdU阴性对照是设置为流式细胞仪分析的门控需要，注入额外的年龄，性别和实验匹配编小鼠用生理盐水等体积的无的EdU。这将提供所需的控制（没有的EdU控制）。这种控制应包括为每个研究，以便在试剂批次变化和流式细胞仪成立。 </li></ol> 2.心收获<ol><li>在第 6 次的EdU注射后24小时，用牺牲颈椎脱位（或替代附表1民政厅批准的方法）小鼠。 </li><li>将牺牲动物仰卧位，并从中期腹部手术剪隔膜使皮肤切口。 </li><li>切割膜片，和从体腔保持胸骨远，切断双侧暴露心脏。 </li><li>轻轻抬起心脏并切断流出道的大血管解剖的心脏出了胸腔。 </li><li>立即将每个心脏中含有10ml个体15ml离心管的PBS冷却至4℃。 </li><li>传输心中SEP独的10cm陪替皿，用手术刀切成各两个心脏在矢状方向和用一对镊子轻轻挤压洗心脏。然后更换PBS除去尽可能多的血越好。重复此步骤两次。 </li><li>将每个心脏的两个部分为同一个人的1.5 ml离心管。 </li><li>继续到步骤3或备选存储标本-80℃如在步骤2.9和2.10详述。 </li><li>使用地下25针，使离心管盖了一个小洞，以防止开盖因气体膨胀时，被处理的心脏。 </li><li>标记每个微量离心管中并发生在液氮的容器冻结。 </li></ol> 3.核解离和心肌细胞核标签注:此细胞核分离和心肌细胞核标记是从贝格曼等人的改编11注射所有样品执行此过程。用EdU和生理盐水（无控制的EdU）阴性对照。 表1为执行这些步骤所需的解决方案的良方。 <ol><li>对于要分析的每个心脏，放置36毫升的1％BSA / PBS溶液到超速离心管，温育30分钟，弃去溶液，然后风干。 注意:此管在步骤3.6中使用。 </li><li>将个人心中冻结在上用手术刀让心中在切碎过程中解冻的冰切末10毫米盘。 </li><li>放置剁碎心入15 m​​l的细胞裂解缓冲液在50毫升离心管中，并在室温下以25,000rpm下与探针均化器均化15秒。 </li><li>添加另外15 mL细胞裂解缓冲液的离心管，并转移到取40ml DOUNCE具有大间隙杵。用杵执行10笔，并通过100微米，然后40微米的细胞滤网过滤到50ml离心管中。 </li><li>离心10分钟T对于4℃700 XG，去除上清。 </li><li>重悬在25毫升蔗糖梯度溶液的核颗粒和层中包含的10毫升在3.1编写超速离心管的新鲜蔗糖梯度液上层溶液细胞核。 </li><li>离心机13,000 XG 1小时在4℃使用摆动出管转子。 </li><li>以下离心，弃去上清，重悬在1300微升细胞核存储缓冲器的细胞核沉淀在1.5ml微量离心管中，并标记PCM1。 </li><li>除去300微升该悬浮液到一个新的离心管中，并加入到700微升新鲜晶核存储缓冲液中。标签此管作为ISO控制。 </li><li>添加抗PCM1在8微克/毫升到标PCM1离心管的终浓度，并在8微克/毫升到标ISO控制离心管的浓度添加兔IgG同种型对照抗体。 </li><li>在4℃孵育过夜。继incub通货膨胀，离心机在700×g离心10分钟，弃去上清液，用1ml新鲜细胞核存储缓冲器，悬浮和离心的再次在700×g下10分钟更换。弃去上清液并用1ml新鲜细胞核存储缓冲器的替换。 </li><li>加入1μl的F（ab&#39;）山羊抗兔IgG（H + L）抗体（FITC或等效绿色荧光色素）的2片段来实现的2微克/毫升的最终浓度。 注意:使用的F（ab&#39;）2片段抗体减小为免疫细胞，包括巨噬细胞和B细胞的非特异性标记的潜力。 </li><li>在铝箔包裹管，以从光保护和孵育在4℃下1小时。 </li></ol> 4.掺入的EdU检测<ol><li>稀的10倍浓缩的EdU反应缓冲液1:10与去离子水。 </li><li>离心机在PCM-1，并在700×g离心10分钟，ISO控制标记的细胞核的悬浮液，弃去上清，重悬所述细胞核沉淀在1ml的1％BSA / PBS溶液。洗细胞核沉淀两次。 </li><li>最后一次洗涤后，弃去上清，用100μl的EdU固定液替换。 </li><li>包裹管中铝箔从光以保护并在室温下孵育15分钟。 </li><li>在700 xg离心用1ml的1％BSA / PBS溶液，离心机的洗涤样品两次，并在室温下用1×基于皂苷透化溶液进行15分钟孵育。 </li><li>制备1×的EdU标记混合物（ 表2）。 2反应至少将需要包括没有的EdU的控制。 </li><li>加入500μl1X的EdU标记鸡尾酒直接向已经含有在100μl1×基于皂苷透化溶液的细胞核各样品并在室温下保护光30分钟孵育。 </li><li>离心机在700 xg离心，重悬在1ml的1％BSA / PBS中，并重复此步骤两次。 </li><li>离心机在700 XG，弃去上清液，用4取代00微升DNA染色溶液（见材料表 ）。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂的命名 </td></tr><tr><td> 1.细胞裂解液 </td></tr><tr><td> 0.32M蔗糖</td></tr><tr><td>的10mM的Tris-HCl（pH值= 8） </td></tr><tr><td> 5毫米氯化钙2 </td></tr><tr><td> 5毫米醋酸镁</td></tr><tr><td> 2.0毫摩尔EDTA </td></tr><tr><td> 0.5毫米EGTA </td></tr><tr><td> 1毫摩尔DTT </td></tr><tr><td> 2.Sucrose梯度解决方案 </td></tr><tr><td> 2.1蔗糖</td></tr><tr><td>的10mM的Tris-HCl（pH值= 8） </td></tr><tr><td> 5毫米醋酸镁</td></tr><tr><td> 1毫摩尔DTT </td></tr><tr><td> 3.核存储缓冲区（NSB） </td></tr><tr><td> 0.44蔗糖</td></tr><tr><td>的10mM的Tris-HCl（pH值= 7.2） </td></tr><tr><td> 70毫米氯化钾</td></tr><tr><td> 10毫米氯化镁2 </td></tr><tr><td> 1.5毫米精胺</td></tr></tbody></table> 表1: 核分离缓冲液成分配方在协议第3条（核解离和心肌细胞核标签）中使用的所有缓冲区。 5.流式细胞仪分析注意:如前面所述-7,11,15-孤立核流式细胞仪进行流量。 <ol><li>首先，创建一个描述前向散射（FSC）和侧向散射（SSC），允许核碎片（ 图2A）的歧视情节。 </li><li>创建情节通过绘制4以鉴别从聚集体（ 图2B）单核&#39;，6-二脒基-2-苯基）吲哚（DAPI）区域（3-405 / 450/50 A）与高度（33-405 / 450 / 50-H）。确保塔T中的3-405 / 450/50的信号收集的线性度，让正确判断DNA含量。 </li><li>创建VS SSC描述535分之488/ 30-A中的曲线图（PCM-1）和，以评估在单线人口的PCM-1的表达显示在5.2创建的单峰门唯一的事件。 </li><li>为了建立一个PCM-1正栅极（ 图2C）运行同种型对照样品。运行PCM1少量标记的样品，以验证PCM1表达人口和门控位置。 </li><li>创建描述SSC-A对四百五分之四百○五/ 50- A（检测DAPI）的阴谋。在这个情节，只显示单线PCM1表达核，使用5.3创建的大门。此图来创建一个只包含2N的细胞核（ 图2D）的额外层次大门。 </li><li>创建描述535分之488/ 30-A对1-633 / 660/20-A到允许表达心肌细胞群的PCM1的标签的EdU的鉴定曲线。只显示这是单线，P核<html> CM1 +和2N使用上述门控。 </li><li>从没有控制的EdU心脏运行样品来设置这些EDU正栅极（ 图2E）。创建的EdU +细胞适当的大门。 </li><li>记录和量化单峰，已掺入的EdU PCM-1表达，2N的细胞核。 </li><li>计算这个人口占总单峰，PCM1 +核的百分比。这将提供新心肌细胞核产生的速率，在7天的EdU脉冲期间，由于总心肌细胞核的百分比。 注意:作为DAPI与DNA结合是化学计量的荧光强度正比于DNA的量。确定倍性基于所述四百五十零分之四百零五/ 50-的信号的强度，如前面7所述。 </li><li>为了评估总心肌细胞群内的套数用成立于5.5的情节和基于DNA浓度7门控策略。 </li></ol>/files/ftp_upload/53979/53979fig2.jpg"/&gt; 图2:门控策略，量化心肌掺入的EdU和套数 （A）原子核是由碎片基于前向散射（FSC）和侧向散射（SSC）歧视。 （B）中，创建一个线性栅极和单核人口是基于DAPI标记和405/450/50高度VS区域信号识别。 （C）的荧光门控使心肌细胞核（PCM-1阳性）和非心肌细胞（PCM-1阴性）晶核从心脏组织的分离。 （D）的DAPI信号的强度被用来确定DNA浓度和PCM1 +心肌细胞群的从而细胞核倍性。在小鼠&gt;心肌细胞核的80％是二倍体（2N）。 （E）荧光门使2N的分离，这从2N纳入埃杜（PCM + / EDU + = 0.9％），心肌细胞核，这些都没有纳入心肌的EdU（PCM +埃杜）。所有步骤在协议5.0中详述。从MDX显示示例-在C57 / BL10背景/ Y小鼠<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/53979fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am J Physiol. 272, H220-H226 (1997).</li></ol>

The authors have nothing to disclose.

要准确量化心肌营业额和再生试验必须真正心肌细胞的产生和非生产性的DNA分裂区分。许多研究继续简单地忽略这些非生产性活动，量化心肌细胞增殖仅通过周期室壁运动及细胞周期标记物的表达。迄今为止一个方法，使心肌营业额的准确定量，同时控制这些非生产性活动仍然引经据典。尤其是，它仍然很难占到心肌多倍体这有助于达到〜心肌DNA复制13的65％。因此，以协助在心肌细胞产生的精确量化，我们已经开发出一种协议，它允许新心肌细胞核的速率的健壮量化，同时排除了导致增加的倍性DNA的复制。虽然这个协议可以新的心肌细胞属间不是歧视灰和心肌细胞的双核，它可以快速地使用，并且准确地计算上限心肌代的（占倍）。因此该协议提供了一种筛选工具，以评估在疾病模型中的心肌细胞的产生和多倍体的速率潜在变化或评估治疗剂的潜在效率。一旦新心肌细胞核产生的速率的变化使用该协议确定了随后的研究可用于确定是否此由于在心肌细胞产生心肌细胞的成核数量的变化，如先前2,13,17,18所述。这些包括在脉冲期间的使用组织学定量心肌核动力学的或从的EdU脉冲动物得到在在单个核和多核心肌群体比较的EdU掺入组织切片的分析。 由于心肌成交额的低水平协议采用埃杜多次注射超过7天。这也使心肌细胞的生成所有潜在的细胞来源"追"，并允许累计心肌细胞核代量化超过这个时间段。根据研究的基础上，在此时间范围可被调整以适应心肌生成的预测水平。对的EdU掺入在心肌细胞核的准确定量，必须有与用于检测的PCM-1反应性的二级抗体的核的没有非特异性标记。因此这将是谨慎，以优化该协议的这个方面，尤其是当比其他的第二抗体建议在这个协议是用来承接附加二级抗体滴定实验。这里所描述的协议使用PCM-1的表达来识别心肌细胞核。虽然这是一个既定的心肌细胞的标记，替代标记可用于验证数据;这些包括特异性针对已被确认为心肌细胞的细胞核1部分局部心肌肌钙蛋白T抗体。同样，替代核局部蛋白可用于在比上述心肌细胞的其他核种群确定和量化的EdU掺入。重要的是，所有的心肌细胞核正在积极进行有丝分裂被排除在分析，该DNA合成的命运是未知的，并且可能导致在任一细胞分裂或增加的倍性。 PCM1是在细胞周期因此心肌经历有丝分裂不会被PCM1表达式确定的M期拆卸。此外，在细胞周期的S期的所有核应排除随后的分析。这可以通过用2N人口包括与2N和4N种群之间的DAPI强度高于DAPI强度选通所有的细胞核来实现。 虽然它是日益接受的是，心脏具有正常老化和急性损伤过程中替换心肌细胞的能力，这种潜在的源和程度仍存在争议。此外，心肌周转率不同的报道1,7,20-22。这可能部分由于在精确地确定和量化新的心肌细胞的产生19的困难。迄今为止，大多数研究仅在使用组织学分析的，并通过胞质蛋白质，包括肌节的蛋白，心肌周转和更新2,4,23,24的量化的表达心肌细胞的识别依赖。使用这些方法来检测增殖标记物的表达，或者作为这里证明的，胸苷类似物的掺入极易造成其它心脏细胞类型为心肌细胞的误认。虽然使用3D共焦成像可以帮助缓解这些问题日ESE方法是昂贵且耗时的。有趣的是，这里描述的方案展示了新的心肌细胞的核生成发生在每周0.17％的速度。这是与其它流式细胞术为基础的研究表明高达0.13％5周成交率一致。虽然这是很有诱惑力根据这些数据来推断全年周转率，因为在以往的研究2,5,25,26，作为周转率在动物13的续航时间动态，这是不合适的。 这种方法具有许多潜在的应用包括评估潜在的治疗剂，以提高心肌再生或调查心脏疾病的心肌周转和影响心肌多倍体的速率。

近年来出现了质疑的证据的假设，心脏是终末分化，有丝分裂后的器官1,2的积累。然而，心肌营业额和再生的量化仍然具有挑战性。 在使用标准的免疫组化技术精确识别罕见的心肌产生的困难以及报告3。此外，心肌细胞产生的细胞来源仍然不明朗与由心肌细胞增殖以及由干细胞分化4-6作出贡献的证据。因此，使用其中所需要的心肌祖细胞表型的知识谱系追踪模式是不可能的，并在一个单一的人口，包括心肌细胞增殖的定量，是不合适的。此外心肌细胞具有无有丝分裂为endoreplication的电位（导​​致多倍体车diomyocyte）或在没有胞质（导致双核心肌）7,8的有丝分裂。心肌营业额的准确定量依赖于这些事件和真正的新的心肌细胞生成之间分辨能力。这产生独特的并发症，因为DNA复制和细胞周期蛋白依赖性激酶的表达在心肌细胞不完全证明真正细胞分裂9,10。 为了协助新心肌生成的量化，如由贝格曼等 7,11与长期的新颖方法中所述，我们已经联合建立核隔离技术，和pericentriolar材料1（PCM1），用于心肌细胞核识别的免疫标记DNA标记和倍体分析。 PCM-1是积聚在分​​化的，非循环细胞的核表面上的中心体蛋白质。以前的研究已经表明，对抗体PCM-1特异性标记心肌细胞核7,11，因此PCM1已被用于由多个独立组来识别心肌1,12,13。此外，我们已经表明，PCM1表达映射到肌钙蛋白-CRE转基因小鼠模型14（ 补充图1）遗传标记的心肌细胞核。 这里所描述的协议使得不论该细胞起源（ 图1A和B），而同时排除核苷标签由于从分析（ 图1C和D）多倍体新心肌细胞核产生的在小鼠心脏的精确和灵敏的识别。虽然这种方法不能用于心肌binucleation控制，它允许这是需要的心肌细胞周转的精确定量新心肌细胞核的快速和强大的定量。此外，它提供了一个快速筛选工具，以评估在心肌细胞产生的动态电位变化。 而DNA标记通常涉及将5-溴-2&#39;-脱氧尿苷（BrdU）标记作为胸苷类似物，这里所描述的协议使用，因为它需要一个更迅速更少的处理步骤的5-乙炔基-2&#39;-脱氧尿苷（EDU）基于测定通过量和不需要的DNA进行免疫检测的变性，使它与其他免疫染色的协议，从而增加了该方法的潜在的下游应用相兼容。 <img alt="图1" src="/files/ftp_upload/53979/53979fig1.jpg" /> 图1: 连续脉冲用EdU标签新的心肌细胞，不论其祖细胞 。 （A）的EdU掺入心肌细胞分裂过程中DNA的。增殖心肌人口将resul吨心肌的增加，或更换，因此是生产性的DNA合成（有助于组织维持和修复）。 （B）中的EdU被掺入心脏祖细胞的细胞分裂过程中的DNA。这将在细胞分化为心肌细胞谱系期间被保留。这种干细胞分化也将导致增加心肌细胞的数目，因此，有助于组织维护和修理。 （C）的心肌细胞均经过导致增加的心肌细胞的倍性，这是与心肌细胞肥大和心肌重塑相关的"非生产"DNA复制的潜能，但不替换丢失的心肌细胞。多倍体的方法，从binucleation不同，因为它导致心肌细胞与含有四个或更多组两条同源染色体（&gt; 2N）的单核。 （D）继连续细胞核pulse，该协议描述了PCM1表达心肌细胞核的核分离和鉴定，使双方心肌套数和掺入的EdU量化。 PCM1表达，用流式细胞仪检测的EdU掺入。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/53979fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>0.32 M sucrose</td><td>Sigma</td><td>84100</td><td></td></tr><tr><td>10 mM Tris-HCl (pH = 8)</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>c5086</td><td></td></tr><tr><td>5 mM magnesium acetate</td><td>sigma</td><td>M-5661</td><td></td></tr><tr><td>2.0 mM EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>0.5 mM EGTA</td><td>Sigma</td><td>63779</td><td></td></tr><tr><td>1 mM DTT</td><td>Sigma</td><td>D0632</td><td></td></tr><tr><td>70 mM KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266</td><td></td></tr><tr><td>1.5 mM spermine</td><td>Sigma</td><td>85590</td><td></td></tr><tr><td>Isotype rabbit IgG- ChIP Grade</td><td>abcam</td><td>abc7415</td><td></td></tr><tr><td>Rabbit anti-PCM-1 antibody</td><td>Sigma</td><td>HPA023374</td><td></td></tr><tr><td>Alexa Fluor 488 F(ab&#039;)2 Fragment of Goat Anti-Rabbit IgG (H+L) Antibody&amp;nbsp;</td><td>Life technologies</td><td>A-11070</td><td></td></tr><tr><td>cell strainers&amp;nbsp; 70 &amp;mu;m and 100 &amp;mu;m&amp;nbsp;</td><td>Fisher scientific</td><td>11597522, 11517532</td><td></td></tr><tr><td>Glass dounce (40 ml) and pestle large clearance</td><td>Sigma</td><td>D9188-1SET</td><td></td></tr><tr><td>EdU (5-ethynyl-2&amp;rsquo;-deoxyuridine)</td><td>Life technologies</td><td>A10044</td><td></td></tr><tr><td>Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit</td><td>Life technologies</td><td>C10634</td><td>This kit inlcudes&amp;nbsp; reagents required for section, EdU reaction buffer,&amp;nbsp; EdU fixative, saponin-based permeabilization solution and the reagents required for the EdU labelling cocktail.</td></tr><tr><td>CyStain DNA 2 step kit,</td><td>Sysmex Partec</td><td>05 5005</td><td>This kit inlcudes&amp;nbsp; reagents required for DAPI labelling (DNA staining solution)</td></tr><tr><td>Probe homogeniser, &lt;em&gt;e.g.&lt;/em&gt;, TissueRuptor</td><td>Qiagen</td><td>9001273</td><td></td></tr><tr><td>TissueRuptor Disposable Probes</td><td>Qiagen</td><td>990890</td><td></td></tr><tr><td>ultracentrifuge</td><td>Sorvall&amp;nbsp;</td><td></td><td></td></tr><tr><td>Facscanto II</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Ultracentrifuge Tube, Thinwall, Polypropylene. 38.5 ml, 25 x 89 mm</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A2153</td><td></td></tr></tbody></table>

simultaneous assessment of cardiomyocyte dna synthesis and ploidy: a method to assist quantification of cardiomyocyte regeneration and turnover

虽然公认的事实是心脏有潜力有限再生正常老化过程中损伤后心肌细胞和心肌细胞的营业额水平低发生，这些事件的定量仍然具有挑战性。这部分是由于该方法的稀有和事实，即多个蜂窝源向心肌维修。此外，心肌细胞内的DNA复制往往导致多倍体心肌和很少导致通过细胞分裂新的心肌细胞。为了准确地量化这些过程之间的心肌细胞周转歧视是必不可少的。这里描述的协议采用长期核苷的标签，以标示它已出现的利用核隔离和随后的PCM1免疫标记识别DNA复制和心肌细胞核的结果全部核。一起这允许C的核苷标签的准确和灵敏的识别ardiomyocyte核人口。此外，4&#39;，6-二脒基-2-苯基标签和细胞核倍性的分析，使新的心肌细胞核的从中多倍体中已掺入的核苷细胞核的鉴别。虽然这种方法不能用于心肌binucleation控制，它允许新的心肌细胞核的快速和强大的量化而占多倍体。这种方法具有许多下游应用包括评估潜在的治疗剂，以提高心肌再生或调查心脏疾病的心肌周转和倍性的影响。该技术也与附加的下游免疫组织学技术，允许核苷掺入定量在所有的心脏细胞类型相容。

这种方法允许增加的EdU掺入的量化由于心肌细胞核分裂，同时控制增加多倍体化。使用基于BrdU的测定中，我们以前表明，在哺乳动物心脏响应慢性心肌损失，在杜兴氏肌营养不良的mdx小鼠模型中，用新的心肌细胞再生。我们用在这里描述的进一步验证我们公布调查结果，并证明该协议的效用的方法。按照描述的协议;成人（12周龄） 的mdx和控制BL10小鼠致敏以每天注射1的EdU的7天，先前报道以允许实验组5,13之间静态分析。脉冲心中的免疫组化分析表明PCM1的表达已纳入埃杜（ 图3A）心肌细胞的存在。然而，所获得的数据全光照免疫组化摹方法可以被误解，因为它已纳入的EdU其他细胞类型可以被误判为心肌细胞。这样的一个例子是表现在补充图1A和B。此外免疫组化方法不区分核分裂多倍体化。流式细胞术在这个协议详细的分析流程能够在MDX和对照组小鼠心肌细胞核分裂的快速定量，同时排除其已将的EdU因多倍体细胞。类似于先前公布的数据13，分析结果显示在mdx小鼠心脏增加新心肌细胞核代的比率相比，年龄匹配的对照组（1.2％和0.17％; 图3B和C）。 <img alt="图3" src="/files/ftp_upload/53979/53979fig3.jpg" /> 图3: </strong&gt; 埃杜量化标记的野生型和 MDX 心 心肌细胞 。（A）为40微米MDX心厚的部分采取Z堆叠投影图像。 PCM-1表达心肌细胞核已纳入埃杜（红色）（绿色）。黄色箭头指示的EdU标记的心肌细胞。 I和II表示个人的EdU标记在Z平面显示心肌细胞。细胞核标记用DAPI（蓝色）（B和C）流式细胞术分离的核表示从12-13周龄的野生型（C57 / BL10）和mdx小鼠代表性的图（MDX -对C57 / BL10背景/年 ）。 （B）直方图显示2N和&gt; 2N核之间DAPI强度和歧视。 （C）地块分析显示，当在本协议详细介绍了2N心肌细胞核人口的EdU结合。对于所有小鼠埃杜在1周给药12周的年龄。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/53979fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充图1:PCM-1是用于心肌细胞核的特异性标记物使用协议描述细胞核从通过杂交TNT-CRE小鼠14与ROSA NT / NG CRE敏感报告线16产生的双转基因小鼠中分离。在肌钙蛋白T（TNT）启动子的控制下，将得到的鼠标CRE活化导致与定位于细胞核的GFP不可逆转表达。核标有同型的控制（ISO）显示，以确定GFP表达心肌细胞核人口（530分之488/ 30-A）的能力。具有抗PCM1核标签（由检测二抗633-660 / 20-A）显示PCM1表达与GFP表达cardiomyocy的相关性TE核人口。在这个例子中98.8心肌细胞核所标识的TNT启动子活性，由PCM1抗体标记的％。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/Supplementary_Figure1.tif" target="_blank">请点击此处下载补充如图1所示。</a> 补充图 2: 挑战通过免疫组织化学技术定量心肌细胞更新 （A）的二维图像显示这似乎也标示埃杜（红）二PCM-1表达心肌细胞核（绿色）。黄色箭头表示这似乎是已注册成立的EdU心肌细胞。这是由于PCM1表达，并使用2维成像可视化时的EdU标记的表观共定位。 （ 二 ）该图像的三维投影识别这些EDU标记细胞不是心肌，但非心肌细胞覆盖PCM1表达心肌细胞核。在12周的用于实验的年龄C57 / BL10小鼠。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/Supplementary_Figure2A.tif" target="_blank">请点击此处下载补充图2A。</a> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53979/Supplementary_Figure2B.avi" target="_blank">请点击此处下载补充图2B。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 反应组分 </td><td colspan="2"> 反应数 </td></tr><tr><td> 2 </td><td>五</td></tr><tr><td> PBS </td><td> 875微升</td><td> 2.19毫升</td></tr><tr><td>铜保护剂</td><td> 20微升</td><td> 50微升</td></tr><tr><td>荧光染料吡啶甲基叠氮化</td><td> 5微升</td><td> 12.5微升</td></tr><tr><td>稀释后的反应在4.1缓冲液</td><td> 100微升</td><td> 250微升</td></tr><tr><td>总反应体积</td><td> 1毫升</td><td> 2.5毫升</td></tr></tbody></table> 表2: 鸡尾酒的EdU成分 。食谱的EdU标记鸡尾酒协议第4版（EDU掺入的检测）必需的。

Watch this Scientific Journal Video about Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover at JoVE.com

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

心肌细胞DNA的合成和倍性的同时评价:一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.3K 次观看.  Newcastle University. Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

视频: Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心脏细胞治疗中干细胞 dna 完整性的评估

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

科研

遗传学

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

细胞周期变化的可视化和核测定心肌出生后

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

发育生物学

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

医学

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

生物学

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

小鼠肝细胞核球状估计的高通量原位方法

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

使用hiPSC衍生心肌细胞的缺血性心脏病模型和基于视频的心肌细胞收缩比较

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

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 ...