Name: preparation of complaint matrices for quantifying cellular contraction
Uploaded: 2010-12-14T17:00:00
Description: Watch this Scientific Journal Video about Preparation of Complaint Matrices for Quantifying Cellular Contraction at JoVE.com

我们感谢乌尔里希施瓦茨实验室用于蜂窝牵引力（Sabass 等 ，2008）的量化的计算跟踪软件。这项工作是由宝来惠康事业奖和美国国立卫生研究院主任的先锋奖（DP10D00354）ML Gardel和医学科学家的国家研究服务奖（5 T32 GM07281）到SP冬季的支持。

1。激活盖玻片表面<ol><li>盖玻片（＃1.5，22x40毫米）清洁使用在前面描述的协议（沃特曼斯托勒，1998年）的一系列肥皂和乙醇洗涤清洁和去除灰尘。 </li><li>放置在一个不锈钢架机架盖玻片，这样盖玻片间距不接触。 </li><li>在化学通风柜（丁腈手套和护目镜建议），终浓度为2％，在异丙醇稀释充满力量3 aminopropyltrimethoxysilane（2毫升硅烷/ 100毫升异丙醇），以填补一个正方形的玻璃盘（〜350毫升?...

<ol>
	<li>Damljanovic, V., Lajerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamid+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamid substrates for cell mechanotransduction assays.</a> Biotechniques. 39 (6), 847-851 (2005).</li><li>Engler, A., Bacakova, L. N. e. w. m. a. n., Hategan, C., Griffin, A., M, D. D. i. s. c. h. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+compliance+versus+ligand+in+cell+on+gel+responses.">Substrate compliance versus ligand in cell on gel responses.</a> Biophys J. 86 ((1 Pt 1)), 617-628 (2004).</li><li>Gardel, M. L., Sabass, B., Ji, L., Danuser, G., Schwarz, U. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+stress+in+focal+adhesions+correlates+biphasically+with+actin+retrograde+flow+speed.">Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed.</a> J Cell Biol. 183, 999-1005 (2008).</li><li>Rajagopalan, P., Marganski, W. A., Brown, X. Q., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparison+of+the+spread+area,+contractility,+and+migration+of+balb/c+3T3+fibroblasts+adhered+to+fibronectin-+and+RGD-modified+substrata.">Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata.</a> Biophys J. 87 (4), 2818-2827 (2004).</li><li>Reinhart-King, C. A., Dembo, M., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+and+mechanics+of+endothelial+cell+spreading.">The dynamics and mechanics of endothelial cell spreading.</a> Biophys J. 89, 676-689 (2005).</li><li>Stricker, J., Sabass, B., Schwarz, U. S., Gardel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+traction+force+microscopy+for+micron-sized+focal+adhesions.">Optimization of traction force microscopy for micron-sized focal adhesions.</a> J. Phys: Condensed Matter. 22, 194104-194114 (2010).</li><li>Sabass, B., Gardel, M. L., Waterman, C. M., Schwarz, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+traction+force+microscopy+based+on+experimental+and+computational+advances.">High resolution traction force microscopy based on experimental and computational advances.</a> Biophys J. 94, 207-220 (2008).</li><li>Yeung, T. <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+substrate+stiffness+on+cell+morphology,+cytoskeletal+structure,+and+adhesion.">Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.</a> Cell Motil Cytoskeleton. 60 (1), 24-34 (2005).</li><li>Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule/organelle+motility+assays.">Microtubule/organelle motility assays.</a> Curr Protoc Cell Biol. , 13.1.1-13.1.21 (1998).</li></ol>

这里描述的步骤设置牵引力显微镜（TFM）实验，计算跟踪程序的实施等，2008）（Sabass，可量化与微米级的空间分辨率的细胞力量。为了优化实验方案，关键是形成一个纯粹的和统一的凝胶基板与ECM的配体的镀层均匀。我们将讨论下面潜在的隐患： 非均匀的凝胶表面或眼泪： 在我们的经验，有几个步骤，似乎形成了一个漂亮的制服凝胶的关键。如果?...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>3-aminopropyltrimethyoxysilane</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>28, 177-8</td>
<td>&nbsp;</td>
<tr>
<td>40% Acrylamide</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0140</td>
<td>&nbsp;</td>
<tr>
<td>2% Bis-acrylamide</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP1404</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP 150-20</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium persulfate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP179</td>
<td>&nbsp;</td>
<tr>
<td>40nm fluorescent micro-spheres</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F8789</td>
<td>&nbsp;</td>
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<td>Sulfo-SANPAH</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>22589</td>
<td>&nbsp;</td>
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<td>Confocal imaging chamber (RC-30)</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>64-0320</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip spinner</td>
<td>&nbsp;</td>
<td>Home-built</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Ultraviolet lamp CL1000</td>
<td>&nbsp;</td>
<td>UVP</td>
<td>95-0228-01</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel rack</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72239-04</td>
<td>&nbsp;</td>
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<td>acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid)</td>
<td>&nbsp;</td>
<td>Invitrogen
</td>
<td>A-20770</td>
<td>&nbsp;</td>
<tr>
<td>Hydrazine hydrate</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>225819</td>
<td>&nbsp;</td>
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<td>Sodium meta-periodate</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>20504</td>
<td>&nbsp;</td>
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<td>Isopropanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A416-4</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (#1.5)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940&#x2010;224</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>16120</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X</td>
<td>&nbsp;</td>
<td>SOPUS Products</td>
<td>www.rainx.com</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>64-19-7</td>
<td>&nbsp;</td>		</tbody>
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preparation of complaint matrices for quantifying cellular contraction

调节细胞粘附到细胞外基质（ECM）是细胞迁移和ECM重构是必要的。粘着斑大分子集会，情侣收缩的F -肌动蛋白细胞骨架的ECM。此连接可以穿过细胞膜，细胞内的机械力的传输底层基板。最近的工作表明ECM的力学性能调节粘着和F -肌动蛋白的形态以及众多的生理过程，包括细胞分化，分裂，增殖和迁移。因此，细胞培养基质的使用，已成为越来越普遍的方法，能够精​​确地控制和调节ECM的力学性能。 为了量化在粘着斑贴壁细胞的牵引力量，兼容基板结合高分辨率成像和计算技术中的一个方法被称为牵引力显微镜（TFM）。这种技术依赖于当地的基板变形诱发细胞收缩幅度和方向的测量。高分辨率荧光显微镜观察荧光标记的蛋白质相结合，这是可能的关联骨架组织和重塑与牵引力。 在这里，我们提出了一个详细的实验方案编制二维的兼容矩阵，为创造一个良好的特点，可调谐的机械刚度，这是适合测量细胞收缩的细胞培养基材的目的。这些协议包括聚丙烯酰胺凝胶的制备，ECM蛋白等凝胶涂料，电镀细胞凝胶，和高分辨率的共聚焦显微镜使用灌注室。此外，我们提供了一个代表性的样本数据表明使用列举的水杉协议的蜂窝力量的位置和震级。

Watch this Scientific Journal Video about Preparation of Complaint Matrices for Quantifying Cellular Contraction at JoVE.com

Preparation of Complaint Matrices for Quantifying Cellular Contraction

在这段视频中，我们展示了用于制造兼容，细胞外基质（ECM）适用于细胞培养镀膜基片的实验技术，适合牵引力显微镜观察细胞的行为流脑刚度的影响。

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The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

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