Name: studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels
Uploaded: 2010-08-10T17:00:00
Description: Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

工作是我们的实验室是由美国国立卫生研究院的赞助支持。

制备<ul><li>盖玻片应高压灭菌。 </li><li>应使用无菌蒸馏水或去离子水，准备解决方案和洗涤盖玻片。 </li><li> AC（40％W / V）和双AC（1％W / V）的解决方案是0.2微米的过滤消毒。不久之前使用无菌过滤，准备10％过硫酸铵（APS;100μg/ml水）。每月更换的APS解决方案。 </li><li>不能进行高压灭菌，如3 APTMS，氯仿，戊二醛，NHS的，并SurfaSil化学试剂均保存在分配瓶仅用于水凝胶的制备。 </li><li>凝胶为了达?...

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <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+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. 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凝胶聚合过程的一个关键因素是，以避免气泡的形成，这将使细胞绑定到ECM涂层凝胶本身，而不是玻璃盖玻片。这是可以预防的仔细吹打后震荡和可视化，确保无气泡已成为被困在凝胶的聚合解决方案。我们始终建议准备额外的“反应”盖玻片和凝胶，以确保有足够的实验。 凝胶用PBS冲洗时，应特别注意支付。吸痰过程中必须避免与水凝胶本身，因为它可能被抓住，撕裂的?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
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<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
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<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

组织刚度，是一个重要的细胞功能因素，并组织刚度的变化通常与纤维化，癌症和心血管疾病的1-11的关联。传统的细胞研究细胞功能的生物方法涉及一个刚性基质上培养细胞（塑料餐具或盖玻片），它可以不考虑弹性ECM或流脑组织之间的刚度变化的影响。 在体内组织遵守的条件，在体外模型，我们和其他人使用ECM涂水凝胶。在我们的实验室，是基于水凝胶聚丙烯酰胺可以模仿生物12见过的组织符合。除了3 APTMS孵化用NaOH产生“反应”的盖玻片。戊二醛是用于交叉连接的3 APTMS和聚丙烯酰胺凝胶。用于水凝胶的聚合丙烯酰胺（AC），双丙烯酰胺（BIS - AC），过硫酸铵溶液。 N -羟基琥珀酰亚胺（NHS）是纳入交联的ECM蛋白凝胶交流解决方案。聚合凝胶，凝胶表面涂有流脑的首选，如纤维连接蛋白，vitronectin，胶原蛋白等蛋白质水凝胶的硬度，可以由流变或原子力显微镜（AFM）和由不同的解决方案中的12的交流和/或双AC百分比调整。在这种方式下，下层刚度可以匹配生物组织的刚度，也可使用流变或AFM量化。细胞可以接种这些水凝胶和培养根据所需的实验条件。成像的细胞和分子生物学分析他们的恢复很简单。在这篇文章中，我们定义为那些有&lt;3000帕斯卡与E和僵硬的组织下层/&gt; 20,000帕斯卡的弹性模量（E）软下层。

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

可以仿照下层刚度对细胞功能的影响<em&gt;在体外</em&gt;使用不同的符合标准的聚丙烯酰胺凝胶。

使用丙烯酰胺水凝胶的细胞功能的研究矩阵刚度的影响

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Research

JoVE Journal

Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

科研

生物学

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Techniques to Induce and Quantify Cellular Senescence

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...