Name: a novel method for localizing reporter fluorescent beads near the cell culture surface for traction force microscopy
Uploaded: 2014-09-16T13:00:00
Description: Watch this Scientific Journal Video about A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy at JoVE.com

作者要感谢的跨学科创新计划项目，伊利诺伊大学，授予12035。 SK是由美国国家科学基金会（NSF）资助的格兰特在UIUC 0965918 IGERT：培养下一代的研究人员在细胞和分子力学和生物纳米技术。

制造和官能化变刚度与嵌入的细胞培养表面附近的荧光微球的PA凝胶。 1，功能化的顶级玻璃盖玻片<ol><li>清洁玻璃盖玻片（＃1.0，​​12毫米直径），用肥皂和水洗涤，然后用乙醇除去多余的灰尘。 </li><li>地方玻璃盖玻片Â磨碎的表面（ 即移液管尖端支架）上，使得它们不接触，以便易于与盖玻片相互作用。 </li><li>外套盖的整个表面上滑动和聚-D-赖氨酸（0.1毫克/毫?...

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当使用这种技术时，至关重要的是，在胎圈溶液适当稀释，并且将珠粒根据所要求的直径选择，PA凝胶基体的刚度，和正在探讨在所需实验现象的大小规模。 应该注意，当稀释前官能顶部玻璃盖片的珠液服用。在凝胶表面珠粒之间的间隔可以通过改变胶体溶液的稀释因子来改变。稀释该溶液过多会降低珠耦合到所述盖片的数量，并最终降低了凝胶珠的数量。稀释该溶液太少可...

活细胞与当地环境中的机械作用已普遍被使用的PA凝胶的研究。这些底物依赖于德姆博和王1997年1，其中之一衬底的主要优点建立一个简单的，良好表征的协议是，它们的刚度可通过改变凝胶溶液的特定成分的浓度进行调整。这提供了一个理想的平台来研究细胞的不同刚度的环境互动。当PA凝胶涂覆有细胞外基质（ECM）蛋白，细胞粘附到它们，从而产生动力。作为细胞力的结果，所述凝胶变形作为弹性体。该变形取决于由细胞和凝胶的弹性性能所施加的力的大小。各种研究都采用PA凝胶研究细胞牵引力。 在PA的凝胶制造一种变体中，荧光微球（珠）被嵌入吨hroughout凝胶定量细胞牵引力上刚性不同2的凝胶。当细胞施力，珠取代从以下凝胶变形的初始位置。变形场是从个体压边位移的测量。这个变形场是利用具有弹性的理论和凝胶来计算牵引力的弹性性能。这些测量提供洞察力，如何细胞的机械感，并与他们的局部微环境3交互。 在许多广泛使用的PA硅胶制作协议，将珠混杂在整个液态，未聚合状态的PA凝胶。一个完全聚合的PA凝胶含有在其整个体积荧光珠。当计算单元的牵引力，珠粒最凝胶表面（细胞 - 基底界面）附近进行监视。这些珠的位移被假设为发生在细胞培养表面为简单起见，在该力计算中ñ。的凝胶的深度范围内的珠粒的实际位置被忽略。然而，在弹性介质（如PA凝胶），珠接近的点的力的应用将移动超过胎圈即进一步远离点。因此，治疗的一个点的位移（在胎圈的位置）从表面作为在蜂窝牵引力的低估的表面结果远侧。错误的程度取决于从表面的胎圈之间的距离。不能在没有加强筋的位置的知识，估计误差。 需要有一个简单的方法来限制​​珠非常接近的细胞培养表面已经解决了一些技巧。一种方法是增加的小珠的密度在整个凝胶，使得有足够数量的小珠在顶端焦平面测量表面非常接近运动。另一项技术涉及建筑共聚焦成像室活细胞成像，使得从光仅在最上面的焦平面珠被收集4。不同的方法，包括覆盖一层极薄包含在已经聚合凝胶珠顶无珠5个 PA凝胶。每一个这些技术中的一个缺点是，在小珠的凝胶内的精确位置是未知的。这引入了误差成珠的位移场，因此细胞力的计算的计算。另一种技术涉及的珠偶联到用磺基SANPAH 6的已聚合的PA凝胶的顶部表面。这种技术保证了珠确实只对PA的凝胶的顶部，但是它们所嵌入在凝胶中的深度的程度是未知的。这可能会创造一个局部地形的细胞，这可能会改变细胞的行为，因为以前的工作已经表明，细胞可以感知力几微米的距离7。最近，图案PA凝胶，1微米直径为f的技巧在正规化阵列luorescent纤连蛋白点标记成立8。在这种情况下，荧光标记物的深度是已知的，并且基本上是零，如纤连蛋白图案被间接地印刷在凝胶表面上。然而，这种方法不能提供连续的环境在其上的细胞可以附着，作为ECM的蛋白被限制为1微米直径的圆点。一种充分整合追踪珠PA凝胶内，并限制他们到一个已知位置的表面非常接近的方法还有待建立。 在这里，我们开发了一种技术，亚微米到微米直径的荧光珠限制为焦平面非常接近的PA凝胶内的细胞培养表面。凝胶通常是固化夹在2片玻璃板之间的未聚合的液体凝胶溶液。一板的被官能化，使得该凝胶牢固地粘附到它。另一种是未官能化的凝胶固化后除去。我们修改这个可移动的玻璃苏通过用一层珠涂布它rface。在夹着官能和胎圈包被的玻璃表面之间的液体凝胶，珠转移到凝胶，同时它被固化。这限制了小珠“一体化的距离进入内1.6微米的表面的凝胶。玻璃底培养皿被用作在其上的凝胶进行固化的粘合表面。以形成聚合期间平顶凝胶表面，圆形盖玻片用于夹心与玻璃底培养皿中的凝胶。之前的凝胶制造，顶部玻璃盖片上涂有聚-D-赖氨酸（PDL），产生一个正的表面电荷。 PDL中与压缩空气吹走，并且珠在水中的溶液被沉积在盖玻片上。我们利用羧化荧光微球，其带有负电荷，并与通过用PDL创建的带正电荷的表面相互作用。用压缩空气，单层吹珠溶液脱盖玻片后珠保持静电耦合到干盖玻片。 PDL中涂层不影响玻璃的凝胶表面的胶粘性，因为载玻片未损坏并从PA凝胶充分完整移除。 在玻璃底培养皿是由粘附通过治疗97％3-氨丙基trimethoxysliane和0.5％戊二醛。所需的刚性PA的凝胶是由通过标准程序9混合适当的双丙烯酰胺和丙烯酰胺的含量产生。凝胶溶液的液滴被吸至玻璃底培养皿中。含珠的玻璃盖片是用来夹持与培养皿的凝胶。当凝胶固化时，顶盖滑动除去留下嵌入在PA的凝胶内为1.6μm的表面上的小珠。

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			<td height="21" style="height:21px;width:255px;">Name</td>
			<td style="width:128px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">97% 3-Aminopropyl-trimethoxysliane (APTES)</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Glutaraldehyde</td>
			<td>Polysciences, Inc.</td>
			<td>111-30-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1M HEPES buffer solution</td>
			<td>Sigma-Aldrich</td>
			<td>83264</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">40% Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">2% Bisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M1533</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">0.1 um Fluorescent microspheres (mCherry)</td>
			<td>Invitrogen</td>
			<td>F8801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fibronectin, Human, 1mg</td>
			<td>BD Biosciences</td>
			<td>354008</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ammonium Persulfate</td>
			<td>Bio-RAD</td>
			<td>161-0700</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tetramethylethylenediamine (TEMED)</td>
			<td>Bio-RAD</td>
			<td>161-0801</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Poly-D-Lysine</td>
			<td>Millipore</td>
			<td>A-003-E</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)&#160;</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">N-hydroxysulfosuccinimide (NHS)</td>
			<td>Thermo Scientific</td>
			<td>24500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">.05% Trypsin-EDTA (1X)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NaCL</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acrylic Acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>147230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7757</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">35 mm Glass bottom dish with 14 mm micro-well #1 cover glass</td>
			<td>In Vitro Scientific</td>
			<td>D35-14-1-N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glass cover slips, 12 mm diam.</td>
			<td>Ted Pella, Inc.</td>
			<td>26023</td>
		</tr>
	</tbody>
</table>

a novel method for localizing reporter fluorescent beads near the cell culture surface for traction force microscopy

PA的凝胶已经长期被用作研究，由于容易制造和能力调整自己的弹性性质的细胞牵引力的平台。当基片涂覆有细胞外基质蛋白，细胞粘附在凝胶并施加力，使所述凝胶变形。变形取决于细胞的牵引力和凝胶的弹性性能。如果该表面的变形场是已知的，表面的牵引力可使用弹性理论计算。凝胶变形被嵌入荧光标记物珠粒均匀地进入凝胶常用的测量。探针置换的凝胶变形。凝胶的表面附近的探针被跟踪。报告的这些探头的位移被认为是表面的位移。从表面的深度将被忽略。这种假设在介绍牵引力评估错误。用于细胞力精确的测量，重要的是对于被称为珠的位置。我们已经开发一种技术，其利用简单的化学反应来限制荧光标记珠，0.1和1微米的直径，在PA的凝胶，在1.6微米的表面。我们涂上聚-D-赖氨酸（PDL）和荧光珠盖玻片。 PA凝胶溶液，然后在盖玻片和粘附表面之间。荧光珠在固化过程中转移到凝胶溶液。聚合后，在PA凝胶包含在一个平面上接近凝胶表面荧光珠。

共焦成像被用来确定该珠确实凝胶表面的下方，并在凝胶深度内量化它们的精确位置。一个不同的波长比所述凝胶内的荧光珠沉降的表面上，并嵌入荧光纳米粒子与那些在表面上的距离，使用质心确定算法来计算。在凝胶表面上的加强筋的位置用作用于凝胶，其中细胞在粘附施加力的表面的顶表面上的参考。在两种情况考虑（与红色小珠的表面上，反之亦然凝胶绿色珠）的示意图如图3</stron...

Watch this Scientific Journal Video about A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy at JoVE.com

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

制造含有荧光探针聚丙烯酰胺（PA）的凝胶传统技术涉及的夹附着表面和玻璃载片之间的硅胶。这里，我们表明，涂敷该滑动用聚-D-赖氨酸（PDL）和荧光探针的局部化的探针内1.6微米的凝胶表面。

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 ...

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

科研

生物工程

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

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. ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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 ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...