Name: isolation, culture, and functional characterization of adult mouse cardiomyoctyes
Uploaded: 2013-09-24T18:10:00
Description: Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

1。心肌细胞分离 材料（图1） Microdissecting钳 组织钳 细腻的止血钳 止血钳 Microdissecting，锯齿，弯钳 经营剪刀，直 经营剪刀，弯 15毫升Falcon管（5） 60毫米培养皿 磷酸盐缓冲盐水（PBS）中 尼龙网 - 400微米孔径 小漏斗 涂蜡，编织丝绸4-0 19毫米，长7-10厘米 非注射针，钝端，24 G或商业动物喂?...

<ol>
	<li>Chlopcikova, S., Psotov&#225;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+cardiomyocytes-a+model+for+the+study+of+morphological,+biochemical+and+electrophysiological+characteristics+of+the+heart.">Neonatal rat cardiomyocytes-a model for the study of morphological, biochemical and electrophysiological characteristics of the heart.</a> Biomedical Papers. , (2001).</li><li>Rosenthal, N., Brown, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+ascending:+perspectives+for+human-disease+models.">The mouse ascending: perspectives for human-disease models.</a> Nat. Cell Biol. 9, 993-999 (2007).</li><li>Ballou, L. M., Lin, R. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+and+mTOR+kinase+inhibitors.">Rapamycin and mTOR kinase inhibitors.</a> J. Chem. Biol. 1, 27-36 (2008).</li><li>Brette, F., Orchard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Tubule+Function+in+Mammalian+Cardiac+Myocytes.">T-Tubule Function in Mammalian Cardiac Myocytes.</a> Circ Res. , (2003).</li><li>Sakata, Y., Hoit, B., Liggett, S., Walsh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decompensation+of+pressure-overload+hypertrophy+in+G?q-overexpressing+mice.">Decompensation of pressure-overload hypertrophy in G?q-overexpressing mice.</a> Circulation. , (1998).</li><li>Lieu, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+Transverse+Tubules+Contributes+to+Non-Uniform+Ca+2+Wavefronts+in+Mouse+and+Human+Embryonic+Stem+Cell-Derived+Cardiomyocytes.">Absence of Transverse Tubules Contributes to Non-Uniform Ca 2+Wavefronts in Mouse and Human Embryonic Stem Cell-Derived Cardiomyocytes.</a> Stem Cells and Development. 18, 1493-1500 (2009).</li><li>Lim, H., De Windt, L., Mante, J., Kimball, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+cardiac+hypertrophy+in+transgenic+disease+models+by+calcineurin+inhibition.">Reversal of cardiac hypertrophy in transgenic disease models by calcineurin inhibition.</a> J. Mol. Cell Cardiol. 32 (4), 697-709 (2000).</li><li>Muthuchamy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+a+familial+hypertrophic+cardiomyopathy+mutation+in+alpha-tropomyosin+manifests+cardiac+dysfunction.">Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.</a> Circ. Res. 85, 47-56 (1999).</li><li>M&#252;ller, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+the+cardiac+sodium+calcium+exchanger+promoter+in+adult+and+neonatal+cardiomyocytes+by+Nkx2.5+and+serum+response+factor.">Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor.</a> J. Mol. Cell. Cardiol. 34, 807-821 (2002).</li><li>Epstein, F., Hunter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+for+cardiac+hypertrophy+and+failure.">Signaling pathways for cardiac hypertrophy and failure.</a> New England Journal of Medicine. 341 (17), 1276-1283 (1999).</li><li>Snopko, R., Aromolaran, A., Karko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+modifies+Ca+2++signaling+during+excitation-contraction+coupling+in+neonate+cardiac+myocytes.">Cell culture modifies Ca 2+ signaling during excitation-contraction coupling in neonate cardiac myocytes.</a> Cell Calcium. , (2007).</li><li>Ranu, H., Terracciano, C., Davia, K. <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+Na+/Ca2+-exchanger+overexpression+on+excitation-contraction+coupling+in+adult+rabbit+ventricular+myocytes.">Effects of Na+/Ca2+-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes.</a> J. Mol. Cell Cardiol. 34 (4), 389-400 (2002).</li><li>Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P. H., Kass, D. A., Marban, E., Tomaselli, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+mechanism+of+action+potential+prolongation+in+ventricular+myocytes+from+dogs+with+pacing-induced+heart+failure.">Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure.</a> Circ. Res. 78 (2), (1996).</li><li>Splawski, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+of+SCN5A+sodium+channel+implicated+in+risk+of+cardiac+arrhythmia.">Variant of SCN5A sodium channel implicated in risk of cardiac arrhythmia.</a> Science. 297, 1333-1336 (2002).</li><li>Zhou, Y., Wang, S., Zhu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+adenoviral+infection+of+adult+mouse+cardiac+myocytes:+methods+for+cellular+genetic+physiology.">Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology.</a> Am. J. Physiol. Heart Circ. Physiol. 279 (1), 429-436 (2000).</li><li>Sussman, M., Lim, H., Gude, N., Taigen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+cardiac+hypertrophy+in+mice+by+calcineurin+inhibition.">Prevention of cardiac hypertrophy in mice by calcineurin inhibition.</a> Science. , (1998).</li><li>Heinzel, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+diazoxide-induced+formation+of+reactive+oxygen+species+and+loss+of+cardioprotection+in+connexin+43+deficient+mice.">Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice.</a> Circ. Res. 97, 583-586 (2005).</li><li>Li, X., Heinzel, F. R., Boengler, K., Schulz, R., Heusch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+connexin+43+in+ischemic+preconditioning+does+not+involve+intercellular+communication+through+gap+junctions.">Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions.</a> J. Mol. Cell. Cardiol. 36, 161-163 (2004).</li><li>Rockman, H. A., Ross, R. S., Harris, A. N., Knowlton, K. U., Steinhelper, M. E., Field, L. J., Ross, J., Chein, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+atrial-specific+and+inducible+expression+of+an+atrial+natriuretic+factor+transgene+in+an+in+vivo+murine+model+of+cardiac+hypertrophy.">Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.</a> Proc. Natl. Acad. Sci. USA. 88, 8277-8281 (1991).</li><li>Nakamura, A., Rokosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LV+systolic+performance+improves+with+development+of+hypertrophy+after+transverse+aortic+constriction+in+mice.">LV systolic performance improves with development of hypertrophy after transverse aortic constriction in mice.</a> Am. J. Physiol. Heart Circ. Physiol. 281 (3), 1104-1112 (2001).</li><li>Bostr&#246;m, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PGC1-?-dependent+myokine+that+drives+brown-fat-like+development+of+white+fat+and+thermogenesis.">A PGC1-?-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.</a> Nature. 481, 463-468 (2012).</li><li>Liao, R., Jain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+functional+analysis+of+adult+mouse+cardiomyocytes.">Isolation, culture, and functional analysis of adult mouse cardiomyocytes.</a> Methods Mol. Med. 139, 251-262 (2007).</li><li>Volz, A., Piper, H. M., Siegmund, B., Schwartz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+of+adult+ventricular+rat+heart+muscle+cells+in+serum-free+primary+culture.">Longevity of adult ventricular rat heart muscle cells in serum-free primary culture.</a> J. Mol. Cell. Cardiol. 23, 161-173 (1991).</li><li>Guatimosim, S., Guatimosim, C., Song, L. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 689, 205-214 (2010).</li><li>Yang, J., Delaloye, K., Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+and+Perfusion+Techniques+for+Studying+Ion+Channels+Expressed+in+Xenopus+oocytes.">Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes.</a> J. Vis. Exp. (47), e2269 (2011).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recording+of+Ion+Channels+Expressed+in+Xenopus+Oocytes.">Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes.</a> J. Vis. Exp. (20), e936 (2008).</li><li>Arman, A. C., Sampath, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recordings+from+Mouse+Retinal+Neurons+in+a+Dark-adapted+Slice+Preparation.">Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation.</a> J. Vis. Exp. (43), e2107 (2010).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+patch+clamp+recordings+from+motor-neuron+and+target+skeletal+muscle+in+zebrafish.">Paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish.</a> J. Vis. Exp. (45), e2351 (2010).</li><li>Maier, S., Westenbroek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unexpected+role+for+brain-type+sodium+channels+in+coupling+of+cell+surface+depolarization+to+contraction+in+the+heart.">An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (6), 4073-4078 (2002).</li><li>Shroff, S. G., Saner, D. R., Lal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+micromechanical+properties+of+cultured+rat+atrial+myocytes+measured+by+atomic+force+microscopy.">Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy.</a> Am. J. Physiol. 269, 286-292 (1995).</li><li>Domke, J., Parak, W. J., George, M., Gaub, H. E., Radmacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+mechanical+pulse+of+single+cardiomyocytes+with+the+atomic+force+microscope.">Mapping the mechanical pulse of single cardiomyocytes with the atomic force microscope.</a> European Biophysics Journal. 28, 179-186 (1999).</li><li>Smith, B. A., Tolloczko, B., Martin, J. G., Gr&#252;tter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Viscoelastic+Behavior+of+Cultured+Airway+Smooth+Muscle+Cells+with+Atomic+Force+Microscopy:+Stiffening+Induced+by+Contractile+Agonist.">Probing the Viscoelastic Behavior of Cultured Airway Smooth Muscle Cells with Atomic Force Microscopy: Stiffening Induced by Contractile Agonist.</a> Biophysical Journal. 88, 2994-3007 (2005).</li></ol>

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

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

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

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

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

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

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

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

分离，培养和成年小鼠Cardiomyoctyes的功能分析

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

Research

JoVE Journal

Biology

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

成人从人体皮肤的上皮干细胞的分离和培养

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

Isolation and Culture of Neonatal Mouse Cardiomyocytes

分离新生小鼠心肌文化和

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

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

从成年小鼠切牙牙源性上皮干细胞的分离和培养

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

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

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

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

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

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 ...