作者要感谢在本议定书的拍摄技术援助C. Zambataro。我们非常感谢为弥敦道冲击中心奖葛兰基金会的医学研究（SM），希尔布鲁姆的基础，和国立卫生研究院（P30AG025708）和PO1AG025901支持。 JMF的支持T32AG000266关于老龄问题的巴克研究所的研究授予。

1。第1步 - 心肌隔离<ol><li>准备消化缓冲如下： 10 ×消化缓冲液（超纯水的H 2 O ） <table border="1"><tbody><tr><td></td><td> 终浓度。 </td><td> g / L的 </td><td> g/500 mL的 </td><td> g / L的10倍的解决方案 </td></tr><tr><td>氯化钠</td><td> 130毫米</td><td> 7.597 </td><td> 3.3 </td><td> 75.97 </td></tr><tr><td>氯化钾</td><td> 5毫米</td><td> 0.4 </td><td> 0.2 </td><td> 4.0 </td></tr><tr><td>丙酮酸</td><td> 3纳米</td><td> 0.33 </td><td> 0.165 </td><td> 3.30 </td></tr><tr><td> HEPES </td><td> 25毫米</td><td> 5.96 </td><td> 2.85 </td><td> 59.6 </td></tr><tr><td> MgCl 2的 </td><td> 0.5毫米</td><td> 0.101 </td><td> 0.05 </td><td> 1.01 </td></tr><tr><td>的NaH 2宝4monobasic </td><td> 0.33毫米</td><td> 0.04 </td><td> 0.02 </td><td> 0.40 </td></tr><tr><td>葡萄糖</td><td> 22毫米</td><td> 3.96 </td><td> 1.98 </td><td> 39.6 </td></tr></tbody></table> 7.4用NaOH的pH缓冲</li><li>使用10倍的消化缓冲，使每个将被灌注的心脏（ 在超纯水的H 2 O）以下四种解决方案： </li></ol> 消化缓冲（三本解决方案等分） <ul><li> 50毫升 - 1X消化缓冲区，其中包含： <ul><li> 75微升 - EGTA溶液（100毫米EGTA） </li></ul></li></ul> 消化缓冲液B（其中之一） <ul><li> 25毫升 - 1X消化缓冲区，其中包含： <ul><li> 1.25微升- 氯化钙溶液（1 M） </li><li> 1毫克 - 蛋白酶</li><li> 25毫克 - 胶原酶（CAN还使用75％这一数额= 18.75毫克）** </li></ul></li></ul> 收集缓冲区（其中之一） <ul><li> 2.5毫升 - 1个消化缓冲区，其中包含： <ul><li> 1.25微升- 氯化钙溶液（1 M） </li><li> 0.5毫克 - 蛋白酶</li><li> 15毫克 - 胶原酶（75％= 11.25毫克）** </li><li> 125毫克 - 牛血清白蛋白</li></ul></li></ul> 中和洗涤缓冲液（其中之一 ） <ul><li> 25毫升 - 1X消化缓冲区，其中包含： <ul><li> 6.25微升- 氯化钙溶液（1 M） </li><li> 250毫克牛血清白蛋白</li></ul></li></ul> **胶原酶的来源和一批不同，酶的活性，可能会有所不同 。 它可能是必要的优化添加到这个缓冲区，以防止过 ​​度消化的酶量。 <ol start="3"><li>如以前在D设立了隔离制度etail三个15毫升烧杯消化缓冲1,2上冰，50毫升的消化缓冲管和25 mL的消化缓冲液B准备灌注（消化缓冲液A应循行冲洗至少5毫升，通过该系统的运行），加热水浴中收集的缓冲区，提高温度至37℃，中和洗涤缓冲液在室温下。 </li><li>填写灌注导管和解剖盘的心脏将被清洗和冷消化缓冲A.绑到灌注尖端松散结周围的上部套管缝合（4 - O 6 - O的大小）。这以后将用来保存滑落套管，以防止心脏的主动脉。 </li><li>末期稳重的小鼠腹腔注射麻醉剂（0.25毫升戊巴比妥钠溶液），并确保鼠标是无意识的，对疼痛刺激的反应迟钝。剖开胸腔，沿胸保证金仔细切割膜片（图2）。切优向上反映前肋骨（这些切口被指定为B和A，分别在图2） 的关键-鼠标仍然必须呼吸之前，这些切口。 </li><li>用塑料吸管切成一定尺寸，轻轻地吸进吸管的心脏。切开心脏的胸腔小心离开心脏的主动脉，足以认清主动脉分支（图3）。移液器使用，因为它会导致损害最小的心脏。 </li><li>掉落到一个烧杯冰冷的消化缓冲液A的心脏和血液，冲洗。 15 - 45秒的地方，到第二个烧杯冰冷的消化缓冲的心脏后，一个简单地再次冲洗。在心脏转移到培养皿中也充满冷消化缓冲A. </li><li>盘消化缓冲下一个剥离范围的心脏和仔细分离主动脉及其分支。仔细修剪劣势到最后brancH（图4）。 </li><li>适合进入主动脉导管尖端和6 - O使用阶段设置下解剖镜下缝合结扎与4 - O到位。确保套管不坐太低的主动脉内，仍以上的冠状动脉（图4）。 </li><li>消化缓冲液A通过灌注设置流动，设置放置灌注头，并开始灌注心脏，直到大部分血液从心脏是明确的心脏和离开流体洗净。关键 - 灌注液的温度必须尽可能接近至37 ° C，当它出来的导管。如果解决方案是太热或太冷，然后灌注杀死组织。直到灌注在心脏的时间也很关键。时间窗口必须从清除心脏少于5-10分钟开始，直到灌注。心脏必须拆除在麻醉下，没有CO 2附加或颈椎脱位。 </li><li>一旦血液是从心脏HED，消化缓冲A.切换到消化缓冲液B确保消化缓冲液B现在是通过流经心脏。该解决方案将开始有一个黄色的色调。等待2-3分钟，直到心脏有迹象显示失去它的形状，开始显得圆润，这是一个迹象表明，消化工作。心肌灌注收益应该出现轻。 </li><li>心室成50 ml锥形管含15毫升收集缓冲区，用剪刀和地点，在37 ° C。轻轻切成小块心室同时放入收藏缓冲区组织。轻轻混匀，用塑料吸管解散细胞与细胞间的粘连和一个单细胞的心肌细胞混合。 </li><li>当大多数组织的解散，（不到1分钟），让较大的组织块沉底。删除和保存上清15 mL锥形管。让我们从上清引力球团超过10至20分钟（15分钟的首选）在室温下。 </li><li>取下并丢弃上清液。加入5毫升中和洗涤缓冲颗粒，轻轻混匀细胞重悬于他们清洗沉淀。此时细胞会立即准备为单细胞分析（继续执行步骤2）选择。 </li></ol> 2。第2步 - 单核细胞的分离<ol><li>准备一个使用火焰和玻璃毛细管将用于操纵细胞的显微。显微操作是简单的很细的毛细管，然后附加到一个长的软管可以移动或拿起吸力时的细胞。 </li><li>立即着手选择的单个细胞在显微镜下使用一把拉住吸管和气密舱，以防止材料被转移到单细胞样本样本创建显微（尼康Eclipse的TS100，20X）。确保是健康的细胞群（70％可行的），而不是从隔离过程中损坏。 </li><li>稀释一个小培养皿中的细胞，使他们可以单独选择。采摘细胞时，一定要捕获的细胞体积小（小于2μL）。只选择健康的细胞，具有独特的正常心肌细胞形态（图5）。 </li><li>单个细胞放入单个PCR管底部，并立即冻结干冰。这些细胞，然后保存于-80℃直至他们将被用于基因表达分析。 </li></ol> 3。步骤3A - 单细胞的qPCR基因表达要执行单细胞的qPCR采用从单细胞基因表达他们的Biomark纳流控的qPCR阵列Fluidigm公司开发适应协议（Fluidigm公司-促进发展协议＃30）3,4 。 <ol><li>反转录 - 特定目标扩增（RT - STA）的准备。要杜松子酒，悬浮感兴趣的所有基因的引物对（我们已经使用了125对引物，成功）在100微米（10毫米三，pH值8.0，0.1毫米EDTA）1X DNA悬浮缓冲。合并和进一步淡化正向和反向引物对每个检测20微米。最后，一​​个终浓度为200 nm为每个将在RT - STA反应扩增检测引物对稀释成一个解决方案。要做到这一点，每对引物，并稀释他们1:100。例如，如果你有30对引物检测20微米，你将需要添加1μL每对（30μL总），然后进行70μLDNA悬浮缓冲量的其余部分进入决赛稀释体​​积为100μL。 </li><li>现在，底漆解决方案准备，组装的RT - STA反应预混。准备足够的掌握每一个你想放大的单个细胞混合。 <table border="1"><tbody><tr><td> 试剂 </td><td> μL，卷（每反应） </td></tr><tr><td> CellsDirect 2X反应混合</td><td> 5.0 </td></tr><tr><td>标III RT Plantinum的Taq混合</td><td> 0.2 </td></tr><tr><td> RT - STA底漆的组合（200 nm的每个检测） </td><td> 2.5 </td></tr><tr><td>核酸游离H 2 O（或1X DNA的悬浮缓冲液） </td><td> 1.3 </td></tr><tr><td> 共有 </td><td> 9.0 </td></tr></tbody></table> 表1 RT - STA预混</li><li>立即除去最大相对离心力冷冻细胞和离心30秒，以确保细胞在试管底部定位。加入9μLRT - STA的混合管进行热循环的反应。 <ol class="lalpha"><li> 50℃ - 15分钟</li><li> 95℃ - 2分钟</li><li> 18个周期的<ol class="lroman"><li> 95℃15秒</li><li> 60℃，4分钟</li></ol></li><li>保持在4 ° C </li></ol></li><li>从PCR机器管，加4μLExoSAP - IT的反应。 ExoSAP - 它是一个PCR清理RT - STA的反应，将删除多余的引物和酶试剂。通过下列反应在PCR机器的运行管。 <ol class="lalpha"><li> 37℃ - 15分钟</li><li> 80℃ - 15分钟</li><li>保持在4 ° C </li></ol></li><li>当ExoSAP治疗是完整的;稀释样品与DNA悬挂缓冲1:5。现在准备的qPCR分析样品。 Fluidig​​m的基因表达的qPCR阵列（48.48，或96.96板）一起，我们使用这种材料。它也可以使用这种方法与传统的qPCR仪器仪表（96孔或384孔的格式）。然而，这些仪器通常需要更多的样本输入检测的数量限制，可以是Performed从一个单细胞。 </li></ol> 3。步骤3B - 全转录扩增单细胞和基因芯片提取和扩增<ol><li>使用Sigma的WTA2​​套件（CAT＃WTA2）扩增双链颗粒和单核细胞的cDNA。单细胞通过第一步Sigma的WTA2​​协议，根据制造商的指示“提取”。在这第一步库合成缓冲区的潜伏期在37 ° C 5分钟，扰乱了单细胞的细胞膜和“提取”消息进行放大。 </li><li>单细胞扩增两轮，Sigma - Aldrich的WTA2​​套件的使用方向。执行的库准备步骤，根据制造商的协议，然后再添加库样本的1 / 5到70μLWTA2放大 混合液。放大使用建议的循环参数为25个周期的样本。 </li><li>净化样品USI吴Qiagen公司的QIAquick PCR纯化试剂盒（CAT＃28104）和Qiagen公司的Qiacube进程，使用“清理QIAquick PCR扩增反应标准V4”协议。 </li><li>至5μL浓缩纯化的样品，使用高速真空，并提出通过第二轮的扩增为17个周期（WTA2的扩增，循环参数反复）。 </li><li> Purify的使用QIAquick PCR纯化试剂盒的样品和质量检查，使用NanoDrop分光光度计，安捷伦的生物分析DNA 7500试剂盒，根据制造商的协议。 </li></ol>标签和NimbleGen的基因表达阵列<ol start="6"><li>标签2微克双滞留的cDNA每个扩增的​​细胞样品使用NimbleGen的一种颜色标记试剂盒（CAT＃05223555001）根据制造商的协议。 </li><li> 5微克Cy3标记标记样品杂交12x135k， 小家鼠的NimbleGen基因表达阵列（CAT＃05543797001）ACC奥尔丁制造商的协议。阵列的样品杂交后，幻灯片，然后洗净，干燥，才可以在激光扫描仪成像。 </li><li>使用激光扫描仪扫描的基因表达阵列。在这里，我们使用分子器件100POW设置GenePix 4200A扫描仪，和300 350PMT。然后生成每个阵列使用NimbleGen的NimbleScan软件对文件和随后全转录表达分析每个单细胞阵列（一个典型的稳定表达的心肌成绩单的表是作为补充;表S1）。 </li></ol>如果心脏灌注行之有效应该有一个健康的心脏细胞，保留其典型的矩形形态后隔离的高比例。如果没有进行很好的灌注，然后将有一个大的死细胞百分比（在图5中看到的图像）。如果正确使用WTA2或RT - STA方法的扩增细胞n的高端产品应通过随后的质量控制测试NanoDrop和BioAnalyser（图6）的mRNA样品的质量。对于芯片的工作流程，这是完成后，WTA的扩增的mRNA NanoDrop和生物分析仪测试。为这一分析的代表积极结果如图6所示。 WTA2样品应显示NanoDrop分光光度计读数，以及从生物分析仪（BA）的芯片（图6）电泳读出一个强大的放大。一个稳定，通过单细胞的基因芯片检测的基因表为例（表S1）。对于qPCR的过程中，数据的质量进行评估执行一个在PCR反应，以确保引物扩增所需的产品（图7）年底熔体一步。如果正确的话，这融化的步骤应该产生一个特定的熔融峰。为了说明这一点，纳流控的qPCR数据的一个子集显示在峰值融化吨热图emperature（图8）8。它是可能的，以评估其熔融温度的PCR产物的质量，以确保对非特定产品 5的情况下。这张地图上的每一行代表一个细胞样本（S1 - S40）在43个独立的qPCR实验（A1 - A43号）测试。在这个数字中，很明显，一些分析变化很大，很可能低于PCR的特异性更高更稳定的检测可靠。 <img src="/files/ftp_upload/3302/3302fig1.jpg" alt="图1" /> 图1单细胞的分离和基因表达分析的概述。该程序将展示的鼠标心脏手术切除和单个心肌细胞的隔离。在此过程中所讨论的方法包括：要么全转录扩增（WTA）后的qPCR或微阵列分析的方法。 WTA的过程开始然后Sigma的通用引物结合（二）的mRNA裂解（一）池。扩增的材料，这些引物（C）和扩增（四）阶段的扩展创建微克。然后重复这种放大（五）芯片过程中产生足够的材料。 <img src="/files/ftp_upload/3302/3302fig2.jpg" alt="图2" /> 图2：删除从小鼠的心脏切口位置的代表性。切沿左肋侧壁（一），然后沿左肋下缘（二）削减。切割的上方和下方的心脏血管，允许移除主动脉，同时还留下足够cannulate的心脏。库克6兆焦耳适应的图片。 <img src="/files/ftp_upload/3302/3302fig3.jpg" alt="图3" /> 图3图的胸部鼠标。虚线表示切除的心脏从胸腔切口的位置（一）没有损坏的H<html> eart组织。库克6兆焦耳适应的图片。 <img src="/files/ftp_upload/3302/3302fig4.jpg" alt="图4" /> 图4。主动脉弓及其分支的图像。灰线表示的理想的位置在哪里修剪主动脉（一）。其余的心脏主动脉连接空心套管（二）提示，使主动脉内的适当水平（三）。 F.盖拉德7改编而成的图像。 <img src="/files/ftp_upload/3302/3302fig5.jpg" alt="图5" /> 图5。单个细胞的基因表达分析的选择。每个面板显示在灯光下呈现出典型的心肌细胞形态显微镜成像心肌。健康的心肌细胞是由绿色箭头表示。死亡或濒临死亡的细胞，这些细胞是用红色箭头所示。这些死细胞不应该在分析中使用。 iles/ftp_upload/3302/3302fig6.jpg“ALT =”图6“/&gt; 图6。质量控制WTA的单细胞扩增的结果。 （a）图像的一个NanoDrop分光光度计读数，是适合于从WTA反应扩增成绩单。 （二）从生物分析仪芯片的读数显示从三个细胞扩增的结果。 <img src="/files/ftp_upload/3302/3302fig7.jpg" alt="图7" /> 图7。定量PCR扩增曲线（左图）和峰值熔融温度曲线（右图）。 （一）从质量检测中的表达结果显示，尽管不同的扩增曲线与一个单一的熔融峰。 （二）一个贫穷的引物，有高度可变的熔融峰，这是不理想的表达分析熔融曲线。 <img src="/files/ftp_upload/3302/3302fig8.jpg" alt="图8" /> 图8。质量控制的单细胞定量PCR反应结果离子。此热图的建立是为了显示一种颜色规模的熔融温度。每个定量PCR检测峰的熔融温度（A1 - A43号）40（S1 - 40）的单个细胞。集群分析根据其熔融温度。俯视列每个检测有可能看到的变化融化所有的样品。这个数字表明，一些qPCR实验是非常具体的，而有些则是充满变数，因此不适合单细胞分析。 表S1，这些基因<a href="http://www.jove.com/files/ftp_upload/3302/TableS1_array.csv">微阵列数据的补充表</a> 。列出根据它们在大型数据集的单一心肌细胞检测的鲁棒性。这个基因列表表示通常在单一心肌细胞中表达的基因数目的检测灵敏度。

<ol>
	<li>Wolska, B. M., Solaro, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+adult+mouse+cardiac+myocytes+for+studies+of+contraction+and+microfluorimetry.">Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.</a> Am. J. Physiol. 271 (3 Pt 2), H1250-H1255 (1996).</li><li>Shioya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+isolating+healthy+heart+cells+from+mouse+models.">A simple technique for isolating healthy heart cells from mouse models.</a> J. Physiol. Sci. 57 (6), 327-335 (2007).</li><li>Guo, G., Huss, M., Tong, G. Q., Wang, C., Li Sun, L., Clarke, N. D., Robson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Dev. Cell. 18 (4), 675-685 (2010).</li><li>Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., Wilson, K. D., Leong, D., Rosenberg, J., Yao, M., Robbins, R. C., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J. Clin. Invest. 121 (3), 1217-1221 (2011).</li><li>D'haene, B., Vandesompele, J., Hellemans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+objective+copy+number+profiling+using+real-time+quantitative+PCR.">Accurate and objective copy number profiling using real-time quantitative PCR.</a> Methods. 50 (4), 262-270 (2010).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Anatomy+of+the+Laboratory+Mouse.">The Anatomy of the Laboratory Mouse.</a> Informatics. , (1965).</li><li>Gaillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aorta.">Aorta.</a> Radiopaedia. , (2008).</li><li>St&#229;hlberg, A., Andersson, D., Aurelius, J., Faiz, M., Pekna, M., Kubista, M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+cell+populations+with+single-cell+gene+expression+profiling:+correlations+and+identification+of+astrocyte+subpopulations.">Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.</a> Nucleic Acids Res. 39 (4), e24-e24 (2011).</li><li>Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+processor+for+gene+expression+profiling+of+single+human+embryonic+stem+cells.">A microfluidic processor for gene expression profiling of single human embryonic stem cells.</a> Lab Chip. 8 (1), 68-74 (2008).</li><li>Bahar, R., Hartmann, C. H., Rodriguez, K. A., Denny, A. D., Busuttil, R. A., Dollé, M. E., Calder, R. B., Chisholm, G. B., Pollock, B. H., Klein, C. A., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell-to-cell+variation+in+gene+expression+in+ageing+mouse+heart.">Increased cell-to-cell variation in gene expression in ageing mouse heart.</a> Nature. 441 (7096), 1011-1014 (2006).</li><li>Raj, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature,+nurture,+or+chance:+stochastic+gene+expression+and+its+consequences.">Nature, nurture, or chance: stochastic gene expression and its consequences.</a> Cell. 135 (2), 216-226 (2008).</li><li>Elowitz, M., Levine, A., Siggia, E., Swain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li></ol>

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这种方法有可能产生的功能以及基因表达研究心肌。检查单细胞基因表达分析中的一个新兴领域。研究在单细胞水平的转录水平的优势在于，它允许一个纯粹的细胞群，这是没有可能从整个组织的筹备工作检查。此外，单细胞分析允许为单个细胞中的mRNA水平的随机变异的检查，以定义单元，以前被认为是同质8,9人口。除了 ​​确定的基因有可能在他们的基因表达10,11,12随机，该方法还允许为特定人群的罕见细胞基因表达谱鉴定。 虽然这种方法提供了一个令人兴奋的潜在用途，基因表达分析，有SOM发送警告和注意事项，使用单细胞。单细胞分析的主要限制因素，是在实验中收集足够的细胞，例如方差，实现利益的度量方面的统计意义。在地方利益的组织细胞的数目是不是限制的情况下，可以检查数百个细胞每组，使用方法，如新一代测序，或纳流控阵列。然而，在某些情况下，有可能难以取得足够的细胞从组织利益。这可以部分特质针对性的细胞类型的集合时间密集型程序引起的。然而，精心的策划和单细胞采集出发前准备，可能仍然允许进行严格的单细胞分析与有限的技术性错误。当研究必须考虑的结果，即使隔离细胞的程序已经在众多studie使用S（包括显微镜，电等），隔离本身可能会影响基因的表达。至于用任何方法操纵生物样品，你的调查结果结果应仔细验证，以确保单个细胞中的表达是代表组织本身，而不是技术偏见。如原位杂交的方法可以证明是有用的的，完整的组织，以验证这些结果。最后，关键是要确保生成的数据是经过精心的质量控制检查。如图8所示的数据表明，qPCR实验可能会非常强劲，其特殊性，如法＃13（A13），或有高层次的变化，从而导致检测＃34（A34）的技术，如方差。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>氯化钠</td><td> Sigma - Aldrich公司</td><td> 71378 </td><td></td></tr><tr><td>氯化钾</td><td> Sigma - Aldrich公司</td><td> 60128 </td><td></td></tr><tr><td>丙酮酸</td><td> Sigma - Aldrich公司</td><td> P4562 </td><td></td></tr><tr><td> HEPES </td><td> Sigma - Aldrich公司</td><td> 54457 </td><td></td></tr><tr><td> MgCl 2的 </td><td> Sigma - Aldrich公司</td><td> M1020 </td><td></td></tr><tr><td>的NaH 2 PO 4二氢</td><td> Sigma - Aldrich公司</td><td> P9791 </td><td></td></tr><tr><td>葡萄糖</td><td> Sigma - Aldrich公司</td><td> G6721 </td><td></td></tr><tr><td>氢氧化钠</td><td> Sigma - Aldrich公司</td><td> 72068 </td><td></td></tr><tr><td> EGTA </td><td> Sigma - Aldrich公司</td><td> O3777 </td><td></td></tr><tr><td> 氯化钙 </td><td> Sigma - Aldrich公司</td><td> 21115 </td><td></td></tr><tr><td>蛋白酶，类型第十四</td><td> Sigma - Aldrich公司</td><td> P5147 </td><td></td></tr><tr><td>科亚固氮酶，I型</td><td>沃辛顿</td><td> C9891 </td><td></td></tr><tr><td> 1X DNA悬挂缓冲器（10毫米三，pH值8.0，0.1毫米EDTA） </td><td> TEKnova， </td><td> PN T0221 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> B8667 </td><td></td></tr><tr><td>戊巴比妥钠</td><td>亨利施恩兽医。供应* </td><td>订购供应商联系</td><td> *必须通过兽医或实验动物专业采购</td></tr><tr><td> ExoSAP的IT </td><td> Affymetrix公司/ USB </td><td> 78201 </td><td></td></tr><tr><td> CellsDirect 2X RXN混合</td><td> Invitrogen公司</td><td> 11737-030 </td><td></td></tr><tr><td>标三RT白金的Taq混合</td><td> Invitrogen公司</td><td> 10928-042 </td><td></td></tr><tr><td> RT - STA底漆混合</td><td>内幕交易审裁处</td><td>自定义</td><td></td></tr><tr><td>核酸免费水</td><td> Sigma - Aldrich公司</td><td> W4502 </td><td></td></tr><tr><td> WTA2 </td><td> Sigma - Aldrich公司</td><td> WTA2 - 50rxn </td><td></td></tr><tr><td> Qiaquick PCR纯化试剂盒</td><td> Qiagen公司</td><td> 28104 </td><td></td></tr><tr><td> Qiacube </td><td> Qiagen公司</td><td> 9001292 </td><td></td></tr><tr><td> NanoDrop分光光度计</td><td>NanoDrop </td><td> 2000C </td><td></td></tr><tr><td>生物分析仪的DNA 7500试剂盒</td><td>安捷伦</td><td> 7500套件</td><td></td></tr><tr><td>一种颜色标记试剂盒</td><td>罗氏的NimbleGen的</td><td> 5223555001 </td><td></td></tr><tr><td>小家鼠12x135k阵列</td><td>罗氏的NimbleGen的</td><td> 5543797001 </td><td></td></tr><tr><td> GenePix 4200A扫描仪</td><td>分子器件</td><td> 4200A </td><td></td></tr><tr><td> TransPlex完整的全转录扩增试剂盒（WTA2套件） </td><td> Sigma - Aldrich公司</td><td> WTA2 - 50RXN </td><td></td></tr></tbody></table>

single cell transcriptional profiling of adult mouse cardiomyocytes

虽然许多研究都从心脏组织匀浆研究基因表达的变化，这样可以防止一个组织内的细胞之间的固有的随机变异研究。纯粹通过胶原酶灌注小鼠心脏心肌人口的分离，有利于全转录基因表达的单细胞微阵列的一代，或使用纳流控阵列的具体目标的qPCR。我们在这里描述一个过程来研究单个细胞基因的表达谱，从心脏分离心肌。这种范式允许利益的指标是不依赖于均值（例如一个组织内的细胞之间的差异），这是不可能的基因表达的评价（图1）当使用传统的整个组织工作流程的评价。我们已经取得了强大的扩增双链cDNA的单细胞转录高产微克，有利于我用的芯片ndividual细胞。在此过程中，我们描述的NimbleGen阵列易于使用和定制其设计能力选择使用。另外，逆转录 - 具体目标扩增反应（RT - STA）的，允许数以百计的纳流控PCR扩增目标的qPCR。使用这些方法之一，它是研究细胞之间的变异性的表达，以及表达谱研究从组织内一个罕见的细胞类型。总体而言，单细胞基因表达的方法可以生成的数据，可以潜在地识别配置文件，通常平均在审查从整个组织产生的典型匀浆数以百万计的细胞表达的特异表达。

Watch this Scientific Journal Video about Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes at JoVE.com

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

单细胞的表达谱可以详细的单个细胞的基因表达分析。我们描述为心肌细胞隔离的方法，并编制要么全转录组微阵列或qPCR的具体目标产生的裂解物。

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 ...

single-cell-transcriptional-profiling-of-adult-mouse-cardiomyocytes

Research

JoVE Journal

Biology

17.3K 次观看.  Buck Institute for Research on Aging. 单细胞的表达谱可以详细的单个细胞的基因表达分析。我们描述为心肌细胞隔离的方法，并编制要么全转录组微阵列或qPCR的具体目标产生的裂解物。

视频: Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

科研

生物学

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

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 ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

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 ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

基于滴瓣的成年哺乳动物组织单细胞转录瘤

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

成年小鼠和大鼠心肌细胞的隔离、转染和长期培养

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...