Name: isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy
Uploaded: 2014-05-21T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

这项工作是由国家心脏，肺和血液研究所资助的HL-36573的支持。我们感谢大卫·索瓦先生编辑这篇稿子。

对小鼠的研究都按照程序，美国国立卫生研究院的指导方针进行，而协议已获托莱多大学的机构动物护理和使用委员会，医学与生命科学学院。 1，灌注系统准备<ol><li>隔离的前一天，填充整个灌注系统（包括水库）用快速碱性无残留洗涤剂的溶液。让溶液浸泡到系统中的几个小时或过夜。 </li><li>第二天早上，调节热循环温度至37℃，并让溶液升温。除去洗涤剂溶液并彻底?...

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	<li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li><li>Gross, D. R., Gross, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research. , 109-130 (2009).</li><li>Schl&#252;ter, K. D., Piper, H. M., Dheim, S., Mohr, F. W., Delmar, 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+and+culture+of+adult+ventricular+cardiomyocytes.">Isolation and culture of adult ventricular cardiomyocytes.</a> Practical Methods in Cardiovascular Research. , 557-567 (2005).</li><li>O&#39;Connell, T. D., Ni, Y. G., Lin, K. M., Han, H. P., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+cardiac+myocytes+for+signaling+studies.">Isolation and culture of adult cardiac myocytes for signaling studies.</a> AfCS research reports. 1 (5), 1-9 (2003).</li><li>O&#39;Connell, T. D., Rodrigo, M. C., Simpon, P. C., Vivanco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+cardiac+myocytes.">Isolation and culture of adult mouse cardiac myocytes.</a> Methods in Molecular Biology. 35, 271-296 (2007).</li><li>Nilolskaya, A., Sharma, V., Sigg, D. C., Laizzo, P. S., Xiao, Y. F. <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+models+and+methods.">Cell culture models and methods.</a> Cardiac Electrophysiology Methods and Models. , 213-235 (2010).</li><li>Merx, M. W., Schrader, M., Dhein, S., Mohr, F. W., Delmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+heart.">The working heart.</a> Practical Methods in Cardiovascular Research. , 173-189 (2005).</li><li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression.">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 1667-1674 (2008).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2,3-butanedione+monoxime.">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 262-269 (2012).</li><li>Liu, C. X., 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=Reduction+of+Na/K-ATPase+potentiates+Marinobufagenin-induced+cardiac+dysfunction+and+myocyte+apoptosis.">Reduction of Na/K-ATPase potentiates Marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.</a> J. Biol. 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为了获得最佳的准备，最关键的步骤是：1）及时小鼠心脏挂接到切除后的套管; 2）适当的心脏灌注。另外，还要注意水的质量，灌注温度，pH缓冲液，消毒，化学纯度和洁净，无污染管和室也是重要因素。 18.2MΩ分子生物学级H 2 O的强烈建议缓冲液配制。 200毫摩尔ATP储备溶液的pH值应调整到7.2，这是很容易错过的工序。 关键是要迅速查明主动脉和削减略低于公司第一分公司如由弗林13。...

心肌细胞不增殖。也有一些心房心肌细胞系，如HL-1和AT-1细胞从小鼠心房肿瘤衍生的;然而，没有成年心室的心肌细胞可用于研究线。成年小鼠心肌细胞的原代细胞培养提供了强大的模型心脏研究在细胞和分子水平。迄今为止，它们已被广泛用于生化，生理和药理学研究1。此外，频繁使用转基因小鼠的必要了心肌细胞的分离有效的方法。纯培养允许无相互作用与其他器官的条件和全身循环，例如通过内源性神经激素和激素样因子2,3。然而，心肌细胞的成功分离是具有挑战性的。 我们这里介绍的协议是基于我们与成年大鼠cardiomyocyt经验ES和O&#39;Connell 等人 4,5中记载的方法。尤其是在2007年5，他们描述了从缓冲准备详细的技术，细胞培养和功能测定。自小鼠细胞极易受到挛缩相比，大鼠心肌细胞中，用于灌注，消化，Ca 2 +的耐受性，电镀和培养的小鼠协议的缓冲器或介质补充有非特异性兴奋-收缩偶联酶抑制剂，2， 3 - 丁二酮肟（BDM），以抑制其自发性收缩，因而生存能力和杆状细胞产量显著提高。在这里介绍的协议中，肌细胞是由改性的Langendorff灌注的II型胶原酶分离在从分离的心脏较高的钾缓冲液中。 Ⅱ型胶原酶有效地打破了细胞间基质，并释放细胞。该灌注溶液也保持细胞的新陈代谢处于较低水平6。在additi上，来自哈佛仪器，我们这里使用的离体心脏系统是精心设计的精确的温度和恒压控制7。这种方法提供了高度可重复的制剂和单一的细胞类型，这可用于在培养过夜，用于测量信号蛋白或2-3天培养心肌肥厚测定的均一群体。

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			<td height="41" rowspan="2" style="height:41px;width:257px;">Isolated heart system for small rodent</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">IHSR-mouse 73-4019</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Aortic Cannula, OD 1.0 mm</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">73-2816</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Dumont #4 Forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11294-00</td>
			<td style="width:288px;"></td>
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			<td height="20" style="height:20px;width:257px;">Straight sharp serrated scissors</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">14070-12</td>
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			<td height="20" style="height:20px;width:257px;">Serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11050-10</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Curved, serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11052-10</td>
			<td style="width:288px;"></td>
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			<td height="20" style="height:20px;width:257px;">Straight Mini Serrefines clamps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">18054-28</td>
			<td style="width:288px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:257px;">MEM</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:99px;">11575-032</td>
			<td style="width:288px;"></td>
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			<td height="20" style="height:20px;width:257px;">Collagenase II</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:99px;">4176</td>
			<td style="width:288px;"></td>
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			<td height="21" style="height:21px;">Mucosal universal detergent</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:99px;">Z637181</td>
			<td>Mucasol is a fast alkaline residue-free detergent.</td>
		</tr>
	</tbody>
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isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

技术进步已经使转基因小鼠，包括转基因和基因敲除小鼠，在许多研究领域必不可少的工具。成人的心肌细胞被广泛接受为一个很好的模式心脏细胞生理学与病理生理学，以及医药干预。转基因小鼠排除需要复杂的心肌细胞的感染过程，以产生期望的基因型，这是低效的，由于心肌细胞“终末分化。隔离的高数量和质量心肌功能和文化将极大地有利于心血管疾病的研究，并为细胞信号转导研究和药物开发的重要工具。在这里，我们描述的，可以用很少的培训来实现成年小鼠心肌细胞分离一套行之有效的方法。小鼠心脏被切除，插管到离体心脏系统，然后用无钙及高磷灌注otassium缓冲其次是Ⅱ型胶原酶消化的Langendorff逆行灌注模式。这个协议产生一致的结果为功能性成年小鼠心肌细胞来自各种转基因小鼠的集合。

1，成功的定量分离两个标准用于定量隔离的成功：首先，心肌细胞的分离的总数，和第二，杆状耐钙圆非容错肌细胞的比率。通常这个协议需要大约75-90分钟从心脏切除到心肌细胞电镀和从一个成年小鼠心脏的产量大约100万杆状心肌（共收获细胞70-90％）。这可能与小鼠体重和应变而变化。通常情况下，0.7-1.0万元杆状细胞可以从〜25克C57BL/6J小鼠，从〜35克黑瑞鼠标1.2-1.6万元杆?...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

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

我们描述了成年小鼠心肌细胞分离的可靠方法。该协议得到了一致的结果为从各种转基因小鼠的功能成年心肌文化。

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

Research

JoVE Journal

Biology

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

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞的分离和遗传操作共聚焦成像

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

科研

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

Isolation 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, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

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

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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 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, 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 ...

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 of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...