Name: a simplified system for evaluating cell mechanosensing and durotaxis in vitro
Uploaded: 2015-08-27T15:00:00
Description: Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

这项工作是由美国国立卫生研究院R01 GM47607，CA163296和NSF 1334493至CET支持。我们感谢特纳实验室成员的手稿批判性阅读。本报告中显示的所有数据均来自Wormer 等2014 11转载许可。

1.准备Durotaxis商会<ol><li>为了制备1 6-孔组织培养板，去皮用50毫升锥形管中的平衡。称取约10克，在50毫升试管中的PDMS碱溶液中（该溶液是非常粘稠）。 </li><li>对于90：1的衬底（符合性〜1千帕），通过90分在管与PDMS碱溶液的测定重量来确定的交联剂溶液所需的正确的金额。与PDMS交联剂溶液的计算量添加到同一管中。 例如：10克/ 90 = 0.11克。添加0.11克PDMS交联剂溶液与PDMS碱溶液。 注意：?...

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Q., 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=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. 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在此我们描述了一个简单的实验来研究durotaxis在细胞迁移。此法的主要优势是易于使用准备PDMS中durotaxis室的。基片的刚性可以通过改变碱的PDMS溶液的比率交联剂，以允许在测定中的各种刚性的研究很容易被操纵。然而，该系统中的一个潜在的限制是，细胞仅暴露于基板的刚性的单一变化，而 ​​不是经历由更复杂的系统4提供一个刚性梯度。重要的是，不象聚丙烯酰胺基质中，ECM组分不?...

细胞外基质（ECM）是由结构和交联蛋白，包括胶原蛋白，纤连蛋白和层粘连蛋白的一系列复杂的。虽然它是公认的对ECM提供了细胞组织重要的结构支承，有越来越多的证据表明，细胞积极应对物理变化在其细胞外基质环境来调节多种细胞过程，包括细胞存活，分化和细胞迁移。例如，在ECM的刚性差异能驱动间充质干细胞向不同谱系，具有柔软基材（〜1千帕）促进神经谱系而僵硬（〜25千帕）衬底促进成骨分化1。同样地，增加间质基质刚性已经显示出促进乳腺上皮细胞肿瘤的发生和侵袭到周围组织2,3。 这mechanosign的一个特别有趣的方面阿玲活性导致称为durotaxis，其中细胞优先迁移朝着更刚性基板4,5的处理。细胞不断通过整联蛋白受体感知它们的胞外环境的物理特性结合到ECM。这，反过来，促使许多结构性和信号蛋白的积累到其胞质结构域，以驱动被称为粘着斑或局灶性触点6,7-粘合剂结构的形成。 自整联没有固有的酶活性，信号是从ECM中继通过这些辅助蛋白来协调细胞的反应其变化的环境8。因此，参与调节mechanosignaling和durotaxis关键蛋白质的鉴定和表征是调查的一个重要领域。 各种模型系统已被开发，研究durotaxis 体外 ，但最有利用胶原包被的聚丙烯酰胺基质4。然而，聚丙烯酰胺基片的制备中可以是技术上具有挑战性并在这些试验中使用的胶原必须是化学交联到基底9。聚二甲基硅氧烷（PDMS）基片已被证明表现出的聚丙烯酰胺基片10可比机械性能。然而，PDMS衬底被通过简单地混合碱的比率来制备交联剂和这些基片可以涂有ECM蛋白无需化学交联，从而使PDMS一个更简单的工具，研究刚性对细胞行为的影响。在此，我们介绍如何编写一个简单durotaxis室，其中软PDMS基板集成了硬质玻璃盖玻片。 该法，如下所述，为研究durotaxis一种快速，简便的方法。在这项研究中，我们使用人类U2OS骨肉瘤细胞结合的siRNA介导cdGAP击倒研究在durotaxis 11本黏着蛋白的作用。重要的是，该协议可以很容易地适应个人的要求。其它细胞类型可被取代的U2OS细胞和任何蛋白质可以被击倒或过表达，以确定在durotaxis对细胞行为的影响。此外，该协议可以适于掺入荧光标记的蛋白来分析他们的动态和用FRAP行为或FRET方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

细胞外基质的成分和机械性能是组织类型之间高度可变。此结缔组织基质多样性大大影响细胞的行为，以调节正常和病理过程，包括细胞增殖，分化，粘附信令和定向迁移。在这方面，某些细胞类型的固有能力，以移向称为durotaxis较硬的，或更少的标准矩阵衬底。这种现象扮演胚胎发育，伤口修复和肿瘤细胞入侵期间起重要作用。这里，我们描述一个简单的试验来研究durotaxis， 在体外，用聚二甲基硅氧烷（PDMS）的基板。的描述durotaxis腔室准备创建相对软的PDMS凝胶和刚性玻璃盖玻片之间的刚性的接口。在提供的例子中，我们已经使用了这些durotaxis室展示为CDC42作用/ Rac1的GTP酶激活prote在，cdGAP，在mechanosensing和人类U2OS骨肉瘤细胞durotaxis调节。该测定是易于适应其它类型的细胞和/或利益的其他蛋白质的敲低，探讨在mechanosignaling和durotaxis各自的作用。

所述durotaxis室的示意图示于图1A。软PDMS衬底（90：1的混合物的PDMS碱与交联剂溶液）被铺在6孔皿，玻璃盖玻片放置在PDMS，然后部分地覆盖盖玻片的上表面的顶部上，从而产生一个接口不同的顺应的两个基板之间。软PDMS基板的刚性是〜1千帕，这与脑组织的典型合规性，而玻璃的刚性是大约1-2 GPA 1。所述PDMS和玻璃之间的界面的相衬图像显示由白色箭头在图1B。星号?...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

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