Name: isolation and genetic manipulation of adult cardiac myocytes for confocal imaging
Uploaded: 2009-09-17T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging at JoVE.com

这项工作是由德国国家科学基金会（DFG）和联邦风险评估研究所（BFR，德国）的支持。

对所有的协议，包含四个主要步骤的设计如图1所示。所有步骤都在全面详细描述。细胞分离，转导和文化，由大约24个小时的共焦成像后是一个连续的和强制性的时间表。该病毒的建设应该做超前的细胞的分离和基因转导，然后使用。 1。成年大鼠心脏心肌细胞的分离<ol><li>成年雄性Wistar大鼠重量约250克（6-12周龄）腹腔注射170μL麻醉每100克体重的解决方案anesthetised </li><li>此...

<ol>
	<li>Kaestner, L., Ruppenthal, S., S, ., Licha, K., Lin, C. 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> Molecular Imaging II. Vol. 7370, 737008-737008 (2009).</li><li>Viero, C., Kraushaar, U., Ruppenthal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Cell Calcium. 43 (1), 59-59 (2008).</li><li>Kaestner, L., Lipp, P., Shorte, S. L., Frischknecht, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Cellular and Molecular Biological Functions. , 289-289 (2007).</li><li>Kaestner, L., Lipp, P., Popp, J., von Bally, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optics in Life Science. Vol. 6633, 66330K-66330K (2007).</li><li>Rinne, A., Littwitz, C., Bender, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+delivery+of+short+hairpin+RNA+(shRNA)+mediates+efficient+gene+silencing+in+terminally+differentiated+cardiac+myocytes.">Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.</a> Methods Mol Biol. 515, 107-107 (2009).</li></ol>

鼠心肌细胞的分离描述的过程可以适应其他物种，例如鼠标 4，如有必要。需要适应的一个参数，对消化酶的组合（见下文）和消化的时间。要知道，有些种类（如鼠标）的细胞可能会比其他人（如鼠）更脆弱。 胶原酶的选择可能是在隔离过程中最关键的一步。我们选择Liberase Blendzyme，因为它是一种合成酶的组合几乎没有批到批的变化。传统的方法是使用所谓的原油?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anesthesia solution</td>
<td>&nbsp;</td>
<td>Serumwerk Bernburg GmbH (Ursotamin), Bayer Health Care (Rompun)</td>
<td>&nbsp;</td>
<td>Mixture of 1 ml Ursotamin (100 mg/ml Ketaminhydrochlorid) and 240 &mu;l Rompun (2% Xylazinhydrochlorid)</td>
</tr>
<tr>
<td>Citrate solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>117,64 mg sodium citrate in 10 ml 0.9% NaCl solution</td>
</tr>
<tr>
<td>Solution A</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>134 mM NaCl, 4 mM KCl, 11 mM glucose, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Solution A+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 0.2 mM EGTA, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Liberase solution</td>
<td>&nbsp;</td>
<td>F. Hoffmann&#x2010; La Roche Ltd.</td>
<td>11988476 001</td>
<td>500 &mu;l stock solution in 15 ml solution A (stock solution:
10 mg/ml Liberase Blendzyme 4 in aqua dest.)</td>
</tr>
<tr>
<td>Solution B</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 200 &mu;M CaCl2 and 0.1% DNase solution</td>
</tr>
<tr>
<td>Solution B1/2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1:1 mixture of solution A and solution B</td>
</tr>
<tr>
<td>DNase solution</td>
<td>&nbsp;</td>
<td>Sigma&#x2010; Aldrich Co.</td>
<td>D4527</td>
<td>Deoxyribonuclease I, Type 2 from bovine pancreas, 40 KU (15 mg) are dissolved in 5 ml of 10 mM Tris&#x2010;HCl buffer, adjusted to pH=7.35, additional content: 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol; this solution is mixed with 5 ml glycerol</td>
</tr>
<tr>
<td>Extracellular matrix protein (ECM) solution</td>
<td>&nbsp;</td>
<td>Extracellular matrix protein (ECM) solution</td>
<td>E1270</td>
<td>1 ml stock solution as purchased is diluted with 6 mL
medium M199</td>
</tr>
<tr>
<td>Culture medium M199</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>E15&#x2010;834</td>
<td>Medium M199 supplemented with 1 &mu;l/ml ITS solution and 20 &mu;l/ml penicillin&#x2010;streptomycin solution (5000 units/ml)</td>
</tr>
<tr>
<td>ITS solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>25 mg Insulin, 25 mg Transferrin and 50 &#x03BC;l Selenite stock solution are dissolved in 5 ml aqua dest. (stock solution: 0.5 mg/ml Sodiumselenite in aqua dest.). Add a few drops of 1 M HCl until one should get a clear
solution. Aliquots can be frozen.</td>
</tr>
<tr>
<td>Tyrode</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 2mM MgCl2, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Master mix</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5 &mu;l 10&#215;PCR&#x2010;Buffer, 5 &mu;l MgCl2 (25 mM), 2 &mu;l forward primer, 2 &mu;l reverse primer, 1 &mu;l dNTP&#180;s (10 mM each), 33 &mu;l H2O, 1 &mu;l DMSO, 1 &mu;l Taq&#x2010;DNA polymerase for each sample</td>
</tr>
<tr>
<td>LB&#x2010;Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 g/l Tryptone, 5 g/l Yeast extract, 5 g/l NaCl (and 15 g/l Agar if used as solid medium), pH 7.0</td>
</tr>
<tr>
<td>Pac I</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>R0547L</td>
<td>preparation: 50 &mu;g DNA, 5 &mu;l 10&#215;buffer, 0.5 &mu;l 100&#215;BSA,
0.75 &mu;l Pac I, fill with H2O up to final volume of 50&mu;l</td>
</tr>		</tbody>
		</table>
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isolation and genetic manipulation of adult cardiac myocytes for confocal imaging

从成人心分离的心肌细胞模型某处一侧的胚胎和新生儿的肌肉细胞和其他工作的心脏之间的半路上被广泛接受。因此，心肌细胞，心脏细胞的生理和病理生理的良好模型服务，用于制药的调查，以及转基因动物模型的探索。在这里，我们描述了一种发自内心的细胞分离方法。此外，我们还展示了如何对心肌细胞的基因操纵的培育转基因动物可以不执行：这是长期文化的结合（1周）和腺病毒介导的基因转移。后者是描述，从病毒的建设转导的细胞。它可用于基因编码的生物传感器（GEBs），荧光融合蛋白的表达，同时也为蛋白质的过度表达和向下调节，如利用RNAi。在这里，我们提供一个融合的一个亚细胞结构（高尔基）染色的蛋白质表达例子。这种蛋白的表达，可以通过Z -堆叠的共焦成像的亚细胞结构的三维重建可视化。该协议包括在国家的最先进的细胞培养，分子生物学和生物物理学，从而提供了一个探索新的视野，在蜂窝心脏病的方法。

Watch this Scientific Journal Video about Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging at JoVE.com

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

成人的心肌细胞原代细胞，可以从动物心脏中分离和培养几天。在这个文化时期的腺病毒介导的基因转移，可以用来表达基因编码的生物传感器（GEBs）或荧光融合蛋白。这两种方法都可以通过共聚焦显微镜细胞调查。

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