Name: high throughput traction force microscopy using pdms reveals dose-dependent effects of transforming growth factor-&#946; on the epithelial-to-mesenchymal transition
Uploaded: 2019-06-01T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ÃŽÂ² on the Epithelial-to-Mesenchymal Transi

作者感谢汤姆·科德格、迈克尔·兰德里和克里斯托弗·巴雷特在珠子合成方面给予的帮助。A.J.E. 承认自然科学和工程研究理事会赠款 RGPIN/05843-2014 和 EQPEQ/472339-2015,加拿大卫生研究院赠款 143327 号,加拿大癌症协会赠款号 703930 和加拿大创新基金会项目#32749。克里希南承认国家卫生研究院的拨款R21HL123522 和 R01HL136209。H.Y.得到了魁北克圣贝奇基金会和魁北克自然与技术基金会的支持。作者感谢JohananIdicula在视频和手稿方面给予的帮助,并感谢何子新协助制作视频。

注:以下协议将指导研究人员制造和使用图1所示的多井TFM盘。
1. PDMS硅胶基板的制备
<ol><li>基于两个市售套件的复合混合物制备 PDMS 硅橡胶混合物。<ol>
<li>将 PDMS 套件的 A 部分和 B 部分(例如 GEL-8100,参见材料表)以 1:1 的重量比添加到 50 mL 管中。 注:混合物在旋转器上混合,速度足够慢,混合物在旋转过程中来回流动,以确保完全混合。</li>
		<li...

<ol>
	<li>Harris, A., Wild, P., Stopak, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+rubber+substrata:+a+new+wrinkle+in+the+study+of+cell+locomotion.">Silicone rubber substrata: a new wrinkle in the study of cell locomotion.</a> Science. 208 (4440), 177-179 (1980).</li><li>Oliver, T., Dembo, M., 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=Traction+forces+in+locomoting+cells.">Traction forces in locomoting cells.</a> Cell Motil Cytoskeleton. 31 (3), 225-240 (1995).</li><li>Dembo, M., 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=Stresses+at+the+Cell-to-Substrate+Interface+during+Locomotion+of+Fibroblasts.">Stresses at the Cell-to-Substrate Interface during Locomotion of Fibroblasts.</a> Biophysical Journal. 76 (4), 2307-2316 (1999).</li><li>Yoshie, H., Koushki, N., 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=Traction+Force+Screening+Enabled+by+Compliant+PDMS+Elastomers.">Traction Force Screening Enabled by Compliant PDMS Elastomers.</a> Biophysical Journal. 114 (9), 2194-2199 (2018).</li><li>Park, C. Y., Zhou, E. H., 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=High-throughput+screening+for+modulators+of+cellular+contractile+force.">High-throughput screening for modulators of cellular contractile force.</a> Integrative biology quantitative biosciences from nano to macro. 7 (10), 1318-1324 (2015).</li><li>Kraning-Rush, C. M., Califano, J. P., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+traction+stresses+increase+with+increasing+metastatic+potential.">Cellular traction stresses increase with increasing metastatic potential.</a> PLoS ONE. 7 (2), e32572 (2012).</li><li>Agus, D. B., 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=A+physical+sciences+network+characterization+of+non-tumorigenic+and+metastatic+cells.">A physical sciences network characterization of non-tumorigenic and metastatic cells.</a> Scientific Reports. 3 (1), 1449 (2013).</li><li>Guo, M., Ehrlicher, A. J., 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=Probing+the+Stochastic,+Motor-Driven+Properties+of+the+Cytoplasm+Using+Force+Spectrum+Microscopy.">Probing the Stochastic, Motor-Driven Properties of the Cytoplasm Using Force Spectrum Microscopy.</a> Cell. 158 (4), 822-832 (2014).</li><li>Ngan, E., Northey, J. J., Brown, C. M., Ursini-Siegel, J., Siegel, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+containing+LPP+and+alpha-actinin+mediates+TGFbeta-induced+migration+and+invasion+of+ErbB2-expressing+breast+cancer+cells.">A complex containing LPP and alpha-actinin mediates TGFbeta-induced migration and invasion of ErbB2-expressing breast cancer cells.</a> Journal of Cell Science. 126 (Pt 9), 1981-1991 (2013).</li><li>Klein, S. M., Manoharan, V. N., Pine, D. J., Lange, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monodisperse+PMMA+microspheres+in+nonpolar+solvents+by+dispersion+polymerization+with+a+macromonomeric+stabilizer.">Preparation of monodisperse PMMA microspheres in nonpolar solvents by dispersion polymerization with a macromonomeric stabilizer.</a> Colloid &amp; Polymer Science. 282 (1), 7-13 (2003).</li></ol>

为了这种方法的成功,拥有一个厚度稳定约100μm的均匀涂层样品至关重要。应仔细选择模量,以检查感兴趣的生物系统的物理意义。在制造顶层时,应优化基准荧光粒子的浓度,以便准确分析位移和牵引应力。分析孤立的单个细胞需要比测量连体单层更密集的信任层。此外,基板表面应具有胶原蛋白、纤维蛋白和层宁等粘附分子的稳定和均匀涂层,以确保细胞正确附着在基板表面。安装多井分压器时必须?...

Acto-myosin收缩性是活性细胞力学的基本要素,影响细胞行为从运动和增殖到干细胞分化。在组织中,收缩性驱动从胚胎生成中的极性分离到气道收缩和心脏活动的活动。关键的是,要产生紧张,细胞必须首先粘附在细胞外环境中。这样,这种收缩性就在其周围环境中产生牵引力。牵引力显微镜(TFM)以多种形式出现,作为在不同条件下量化不同细胞的这些力的一种方式。
TFM 领域具有非凡的创新和应用广度,其结果为生物学中包含力学和物理力的新视角铺平了道路。从起皱硅胶基板1开始,研究人员应用了各种技术来测量细胞牵引力。这些方法不断改进,现已达到几微米2的分辨率水平。然而,出现了一个主要问题,那就是难以利用现有的硅胶制造适当低的月面基板。为了规避这一问题,聚丙烯酰胺被采用作为替代品,因为易于在1-20 kPa3的量下创建基板。我们最近在TFM4中实现了非常合规的有机硅,使我们能够制造与聚丙烯酰胺相同的莫杜拉利范围,但具有惰性和强健硅胶的优点。
TFM 方法使有价值的美查诺生物发现得以实现,然而,一个长期存在的缺点是它们的复杂性,通常将其使用限制在工程或物理科学学科的研究人员。这在很大程度上是由于量化收缩性所需的详细校准和具有挑战性的计算。另一个重大挑战是,TFM方法基本上低通量,因此不适合同时研究许多不同的条件或人群。这带来了一个瓶颈,阻碍了TFM从专业生物物理学机构转移到更广泛的生物和药理学应用。
我们最近开发了一种多孔格式的TFM板,使研究人员能够并行化其TFM测量,从而更快地量化收缩性指标,同时探索不同化合物的影响,同时使用更少的试剂4.该方法在各种的美形生物学研究中具有广泛的效用,从评估化合物对细胞活动的影响,到量化分化或疾病的收缩变化。
生物医学研究的一个领域,将大大受益于TFM是研究物理线索如何影响癌细胞的恶性表型。转移,导致90%的癌症相关死亡,其特点是癌细胞离开其原来的肿瘤部位和殖民一个次要部位。细胞要通过组织迁移,进出血管系统,必须从根本上改变其形状,以挤压这些物理屏障,同时产生巨大的力量,沿着细胞外基质拉路或在其他细胞间移动细胞。这些力通过焦点附着力2、3将物质传递到基材上,并可通过TFM进行量化。虽然癌症在生物化学上是异常多样化的,已知突变和蛋白质变化的种类不断扩大,但观察到一些常见的物理变化;在各种癌症中,包括乳腺癌、前列腺癌和肺癌,转移性细胞已被证明能发挥非转移性细胞6、7、8的牵引力2-3倍。这些结果表明,转移性进展与细胞施加的牵引力之间可能存在很强的相关性;然而,合同性的详细时间相关变化很难研究。
上皮到间质过渡(EMT)是一个过程,细胞减少粘附和紧密结介细胞-细胞粘附,变得更加迁移和侵入。除了包括伤口愈合和发育过程在内的生理功能外,EMT 也是转移过程中利用的过程,使其成为研究这一过程的有用模型系统。使用TGF-α,我们可以诱导小鼠乳腺上皮细胞(NMuMG)9中的EMT直接量化这种转化过程中的物理变化,并描述TGF-α对EMT和细胞收缩性的时间和剂量依赖性效应。在本文中,我们通过测量诱导EMT期间契约性的变化来演示此方法的效用。

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			<td class="xl69" height="42" style="height:31.2pt;width:206pt" width="275">GEL-8100</td>
			<td class="xl69" style="width:206pt" width="275">Nusil Technology</td>
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			<td class="xl69" style="width:206pt" width="275">109.6mm&#177; x 72.8mm&#177; x 1mm thickness</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">Target 2TM Nylon Syringe Filter</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">96-well Stripwell Egg Crate Strip Holder</td>
			<td class="xl69" style="width:206pt" width="275">Corning</td>
			<td class="xl69" style="width:206pt" width="275">2572</td>
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			<td class="xl69" style="width:206pt" width="275">3099</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">Ethyl alcohol</td>
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			<td class="xl69" style="width:206pt" width="275">0.95</td>
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			<td class="xl69" height="21" style="height:15.6pt;width:206pt" width="275">2-Propanol</td>
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			<td class="xl69" style="width:206pt" width="275">190764</td>
			<td class="xl69" style="width:206pt" width="275">ACS reagent, &#8805;99.5%</td>
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			<td class="xl66" height="42" style="height:31.2pt;width:206pt" width="275">Fibronectin bovine plasma</td>
			<td class="xl65" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td class="xl65" style="width:206pt" width="275">F1141-1MG</td>
			<td class="xl65" style="width:206pt" width="275">solution, sterile-filtered, BioReagent, suitable for cell culture</td>
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			<td class="xl65" style="width:206pt" width="275">319-005-CL</td>
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			<td class="xl65" height="42" style="height:31.2pt;width:206pt" width="275">DMEM, 1X</td>
			<td class="xl65" style="width:206pt" width="275">Wisent</td>
			<td class="xl65" style="width:206pt" width="275">319-005-CL</td>
			<td class="xl65" style="width:206pt" width="275">4.5g/L glucose, with L-glutamine, sodium pyruvate and phenol red</td>
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			<td class="xl65" height="42" style="height:31.2pt;width:206pt" width="275">FBS (Fetal Bovine Serum)</td>
			<td class="xl65" style="width:206pt" width="275">Wisent</td>
			<td class="xl65" style="width:206pt" width="275">080-150</td>
			<td class="xl66" style="width:206pt" width="275">Premium Quality,&#160;Endotoxin &#60;1, Hemoglobin &#60;25</td>
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			<td class="xl65" style="width:206pt" width="275">330-050-EL</td>
			<td class="xl65" style="width:206pt" width="275">1M, free acid</td>
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			<td class="xl65" style="width:206pt" width="275">511-016-CM</td>
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			<td class="xl65" style="width:206pt" width="275">450-201-EL</td>
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			<td class="xl65" style="width:206pt" width="275">609-065-EL</td>
			<td class="xl65" style="width:206pt" width="275">200mM solution, sterile filtered for cell culture</td>
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			<td class="xl65" style="width:206pt" width="275">450-105-QL</td>
			<td class="xl66" style="width:206pt" width="275">250&#956;g/ml, sterile filtered for cell culture</td>
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			<td class="xl65" height="21" style="height:15.6pt;width:206pt" width="275">Acetic acid</td>
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			<td align="right" class="xl65" style="width:206pt" width="275">537020</td>
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			<td class="xl65" style="width:206pt" width="275">251275&#160;</td>
			<td class="xl65" style="width:206pt" width="275">ACS reagent, &#8805;99.5%</td>
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			<td class="xl64" style="width:206pt" width="275">AC190385000</td>
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			<td class="xl64" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td align="right" class="xl64" style="width:206pt" width="275">221473</td>
			<td class="xl65" style="width:206pt" width="275">ACS reagent, &#8805;85%, pellets</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">TritonX-100</td>
			<td class="xl64" style="width:206pt" width="275">Sigma-Aldrich</td>
			<td class="xl64" style="width:206pt" width="275">X100</td>
			<td class="xl64" style="width:206pt" width="275">laboratory grade</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl76" colspan="4" height="21" style="height:15.6pt;
 width:824pt" width="1100">Bead Synthesis</td>
		</tr>
		<tr height="62" style="height:46.8pt">
			<td class="xl67" height="62" style="height:46.8pt;width:206pt" width="275">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate (DiI)</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">468495-100MG</td>
			<td align="right" class="xl72" style="width:206pt" width="275">97%</td>
		</tr>
		<tr height="42" style="height:31.2pt">
			<td class="xl67" height="42" style="height:31.2pt;width:206pt" width="275">Methyl methacrylate</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">M55909-500ML</td>
			<td class="xl65" style="width:206pt" width="275">contains &#8804;30&#160;ppm MEHQ as inhibitor, 99%</td>
		</tr>
		<tr height="62" style="height:46.8pt">
			<td class="xl67" height="62" style="height:46.8pt;width:206pt" width="275">Inhibitor Remover</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">306312-1EA</td>
			<td class="xl65" style="width:206pt" width="275">Prepacked column for removing hydroquinone and monomethyl ether hydroquinone</td>
		</tr>
		<tr height="42" style="height:31.2pt">
			<td class="xl68" height="42" style="height:31.2pt;width:206pt" width="275">Methacryloxylpropyl Terminated Polydimethylsiloxane</td>
			<td class="xl67" style="width:206pt" width="275">Gelest&#160;</td>
			<td class="xl65" style="width:206pt" width="275">DMS-R31 (25,000g/mol)</td>
			<td class="xl65" style="width:206pt" width="275">Polydimethylsiloxane stabilizer, 25,000g/mol, 1,000 cSt</td>
		</tr>
		<tr height="42" style="height:31.2pt">
			<td class="xl67" height="42" style="height:31.2pt;width:206pt" width="275">2,2&#8242;-Azobis(2-methylpropionitrile) (AIBN)</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">441090-25G</td>
			<td align="right" class="xl72" style="width:206pt" width="275">98%</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl67" height="21" style="height:15.6pt;width:206pt" width="275">Hexane&#160;</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">296090-2L</td>
			<td class="xl65" style="width:206pt" width="275">anhydrous, 95%</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl67" height="21" style="height:15.6pt;width:206pt" width="275">Hexane, mixture of isomers</td>
			<td class="xl67" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">227064-1L</td>
			<td class="xl65" style="width:206pt" width="275">anhydrous, &#8805;99%</td>
		</tr>
		<tr height="42" style="height:31.2pt">
			<td class="xl66" height="42" style="height:31.2pt;width:206pt" width="275">Whatman&#160;qualitative filter paper, Grade 1</td>
			<td class="xl65" style="width:206pt" width="275">Sigma-Aldrich&#160;</td>
			<td class="xl65" style="width:206pt" width="275">WHA1001055</td>
			<td class="xl65" style="width:206pt" width="275">circles, diam. 55&#160;mm,</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl76" colspan="4" height="21" style="height:15.6pt;
 width:824pt" width="1100">Equipment</td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl73" height="21" style="height:15.6pt;width:206pt" width="275">Laurell WS-650Mz-23NPPB</td>
			<td class="xl64" style="width:206pt" width="275">Laurell Technologies</td>
			<td class="xl64" style="width:206pt" width="275"></td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl74" height="21" style="height:15.6pt;width:206pt" width="275">UVP Handheld UV Lamp Model UVGL-58</td>
			<td class="xl64" style="width:206pt" width="275">VWR</td>
			<td class="xl74" style="width:206pt" width="275">21474-622</td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
		<tr height="21" style="height:15.6pt">
			<td class="xl64" height="21" style="height:15.6pt;width:206pt" width="275">Rheometer</td>
			<td class="xl64" style="width:206pt" width="275">Anton Paar</td>
			<td class="xl64" style="width:206pt" width="275">MCR 302 WESP</td>
			<td class="xl71" style="width:206pt" width="275"></td>
		</tr>
	</tbody>
</table>

high throughput traction force microscopy using pdms reveals dose-dependent effects of transforming growth factor-&#946; on the epithelial-to-mesenchymal transition

细胞收缩性在生物学的各个方面都是必不可少的,推动过程从运动和分裂,到组织收缩和机械稳定性,并代表多细胞动物生命的核心元素。在附着细胞中,在细胞在其基质上施加的牵引力中可以看到肌苷收缩。细胞收缩性调节的调节出现在无数的疾病中,使得收缩性成为使用生物物理学作为指标的各种诊断方法中一个有希望的目标。此外,新的治疗策略可以基于纠正细胞收缩性的明显故障。然而,这些应用需要直接量化这些力量。
我们开发了基于硅弹性体牵引力显微镜 (TFM) 的并行多井格式。我们使用硅橡胶,特别是聚二甲基硅氧烷(PDMS),而不是常用的水凝胶聚丙烯酰胺(PAA),使我们能够制造具有不确定保质期的坚固和惰性基材,无需专门的储存条件。与基于柱-PDMS的方法在GPa范围内具有模量不同,此处使用的PDMS非常符合,范围从大约0.4 kPa到100 kPa不等。我们通过将这些大型单片基板在空间上划分为生物化学独立井,为 TFM 创建高通量平台,创建一个多井平台,用于牵引力筛选,与现有的多井系统兼容。
在本手稿中,我们使用此多井牵引力系统来检查到中端过渡 (EMT);我们诱导NMuMG细胞中的EMT,将它们暴露到TGF-β,并量化EMT期间的生物物理变化。我们测量收缩性作为TGF-α暴露的浓度和持续时间的函数。我们在这里的发现证明了并行TFM在疾病生物物理学中的效用。

在添加TGF-β之前,细胞的汇合单层具有类似形状的圆石,并且紧密包装。在TGF-β治疗时,细胞在形态学上变得更长,扩大细胞面积,获得更中位表型。利用用软PDMS弹性体制造的多井装置,研究了细胞在总共17个不同条件下的物理特性。细胞用四种不同的TGF-+浓度(0.5,1,2和4纳克/mL)和4个不同的孵育时间(12,24,48,96小时)进行治疗,这些结果在图5中总结。使用TGF-α处理...

Watch this Scientific Journal Video about High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ÃŽÂ² on the Epithelial-to-Mesenchymal Transi

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ÃƒÅ½Ã‚Â² on the Epithelial-to-Mesenchymal Transition

我们采用硅橡胶 (PDMS) 制造高通量牵引力测定。这种新颖的测定适用于研究各种生物和生物医学过程和疾病中细胞收缩性的物理变化。通过测量上皮到中位过渡期间收缩性增加的TGF-α依赖性增加,我们证明了这种方法的效用。

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

我的实验室对机械生物学感兴趣，它研究细胞如何发挥和受到机械力的影响。我们可以量化机械生物学的一个方法就是测量细胞收缩性。在粘附细胞中，收缩表现在对细胞基板施加的牵引力中。我们使用通常称为牵引力显微镜或 TFM 的方法量化这些力。TFM在各种各样的不同方法中实现，但一个常见的方面是，它是一种相当连续和缓慢的实验室技术，通常在像这里这样的单道菜中完成。这限制了我们一次评估一个人口或条件。在整个细胞培养改善前一段时间，但创建多井菜，我们认为，这不是伟大的，我们可以TFM与相同的增加吞吐量和相同的时间和试剂效率？这是我们的解决方案。因此，我们创造了一个96井牵引力盘。它看起来非常类似于传统的96井组织培养皿，然而，与这道菜，我们不仅可以检查96种不同的细胞培养条件，但也量化其细胞收缩性。这提供了一个比单盘方法更高的吞吐量解决方案，使我们能够检查不同的细胞条件和收缩反应。在癌症转移期间，癌细胞从初级部位传播到二级部位，在我们的体内传播疾病。癌细胞的这种转移行为占癌症相关死亡的90%。上皮癌细胞获得更迁移和侵入性表型的拟议机制之一是上皮-乳质过渡，EMT。在EMT期间，他们变得更加迁移和侵入，改变他们的身体行为。为了更好地了解这些物理行为的变化，我们利用软硅基质测量通过TGF诱导的小鼠乳腺细胞收缩的变化，在这项研究中，我们调查了TGF-影响细胞物理行为的不同浓度和孵化时间。聚二甲基硅氧烷是一种非常合规的橡胶，最低 Young 的模量约为 0.6 千帕。这种材料的刚度也可以使用不同浓度的交联剂（包括 SYLGARD 184.To测量我们使用的流变仪的PDMS的机械性能。混合 PDMS 的 A 部分和 B 部分后，以一对一的重量比加上指定重量百分比的固化剂，将准备的批次装载到流变表板上。确保装载足够的样品以完全覆盖主轴区域。确保主轴完全接触样品，边缘没有空位，这一点很重要。任何额外的样品应仔细修剪，并擦掉板。在0.5%Newsha的振荡剪切应变下进行时间和频率扫描，测量每个样品粘弹性的最终存储模量和时间依赖性。

Research

JoVE Journal

Bioengineering

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Stretching Micropatterned Cells on a PDMS Membrane

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Traction Force Microscopy to Study B Lymphocyte Activation

牵引力显微镜研究B淋巴细胞激活

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...