Name: establishing primary adult fibroblast cultures from rodents
Uploaded: 2010-10-05T17:00:00
Description: Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

<p class="jove_content">我们感谢为我们提供与本议定书​​的第一个版本博士史蒂芬Austad。这项工作是由从美国国立卫生研究院和埃利森医学基金会VG的赠款和支持</p>

<p class="jove_title"> 1。开始之前</p><ol><li>用70％乙醇消毒的剪刀和镊子。 </li><li>置于30 mL烧杯内的小型磁力搅拌器，盖上两层铝箔和高压灭菌。 </li><li>无菌水，准备一个28 Wunsch算法Liberase Blendzyme 3单位/毫升原液。在-20 ° C，0.5毫升分装和冻结解冻之前，每次使用新的等分。解冻后的解决方案可能会出现阴天。旋涡的解决方案，直到它变得清晰。 </li><li>热身细胞培养基。 </li></ol><p class="jove_title"> 2。动物样品制备</p><p c...

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> <em style='font-style: italic'>Cell</em>. <b>7</b>, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> <em style='font-style: italic'>Nat Cell Biol</em>. <b>5</b>, 741-747 (2003).</li></ol>

<p class="jove_content">普通小学的成纤维细胞生物学研究癌症细胞株的建立提供了一个绝佳的替代方案。成纤维细胞的一个重要的优势是，他们不进行癌基因和肿瘤抑制基因的突变，并保持完好的细胞周期检查点。这使得一个首选系统研究了细胞周期调控，DNA修复，和凋亡的正常成纤维细胞。这里所描述的协议提供了一个简单的初级成纤维细胞的分离和维护配方。该协议被用来成功地分离出超过20种啮齿类动物细胞。 <...

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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>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td><strong>Liberase TM Research Grade</strong></td>
<td>&nbsp;</td>
<td>Roche</td>
<td><strong>05401127001</strong></td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

establishing primary adult fibroblast cultures from rodents

<p class="jove_content">在生物学研究中使用的主要细胞，而不是肿瘤细胞株，的重要性是越来越广泛的认可。原代细胞在细胞周期调控，细胞凋亡的研究中的首选，DNA修复，癌细胞进行，在这些过程中所涉及的基因突变。原电池不能无限期由于复制衰老或aneuploidization开始培养。因此，新的文化需要建立定期。啮齿类动物胚胎成纤维细胞的分离过程是公认的，但隔离成年成纤维细胞培养，往往提出了严峻挑战。成人啮齿类动物从人类疾病小鼠模型中分离出的成纤维细胞，可能是来自人类患者的成纤维细胞进行比较时，他们首选的控制。此外，成人的成纤维细胞与怀孕的女性不能轻易获得的野生啮齿类动物工作时，唯一可用的材料。在这里，我们提供了一个从啮齿类动物的皮肤和肺部成年成纤维细胞的分离和文化协议。我们用这个程序成功地隔离开来超过20个啮齿类动物从实验室小鼠和大鼠的成纤维细胞，如河狸，豪猪，松鼠等野生啮齿类动物。 </p>

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents (Video) | JoVE

<p class="jove_content">本文介绍了一种从野生啮齿类动物的皮肤和肺组织的初级成纤维细胞培养的隔离和维护协议。</p>

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