Name: quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy
Uploaded: 2013-02-21T13:00:00
Description: Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

笔者想感谢埃琳娜Lomakina，理查德·Bauserman，玛格丽特杨曼，吉Vaknin，杰西卡·斯奈德，克里斯Striemer，纳库尔Nataraj，红栗橱嗯的，TEJAS Khire，和Eric林对他们的援助，这个项目的。该项目是由美国国立卫生研究院PO1 HL 018208。

1。方法 1.1细胞流动室甲流室， 如图1中所示，构建了使细胞可为1.0帕（10达因/厘米2）的剪切下生长，然后直接转移到一个的庇护MFP3D原子力显微镜（圣巴巴拉，加利福尼亚州）。 <ol><li>流动室，制备实验首先通过清洗载玻片的Piranha溶液（3:1 H 2 SO 4：H 2 O 2）为15分钟，然后用蒸馏水洗涤。然后将它们进行烘烤，以干燥?...

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	<li>Clifford, C., Seah, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+measurement+of+young's+modulus+for+compliant+layers+on+stiffer+substrates+including+the+effect+of+poisson's+ratios.">Nanoindentation measurement of young's modulus for compliant layers on stiffer substrates including the effect of poisson's ratios.</a> Nanotechnology. , (2009).</li><li>Gouverneur, M., Spaan, J. A. E., Pannekoek, H., Fontijn, R. D., Vink, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+stimulates+incorporation+of+hyaluronan+into+endothelial+cell+glycocalyx.">Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx.</a> Am. J. Physiol. Heart. Circ. Physiol. 290 (1), 458-452 (2006).</li><li>Hocde, S. A., Hyrien, O., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+distribution+relative+to+neutrophil+surface+topography+assessed+by+tirfm.">Cell adhesion molecule distribution relative to neutrophil surface topography assessed by tirfm.</a> Biophysical Journal. 97 (1), 379-387 (2009).</li><li>Lipowski, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+as+a+barrier+to+leukocyte+adhesion+and+its+mediation+by+extracellular+proteases.">The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.</a> Annals of biomedical engineering. 40 (4), 840-848 (2012).</li><li>Lu, L., Oswald, S. J., Ngu, H., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+actin+stress+fibers+in+living+cells.">Mechanical properties of actin stress fibers in living cells.</a> Biophysical Journal. 95 (12), 6060-6071 (2008).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Archiv. European Journal of Physiology. 440 (5), 653-666 (2000).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophysical Journal. 87 (6), 4237-4245 (2004).</li><li>vanden Berg, B. M., Vink, H., Spaan, J. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+protects+against+myocardial+edema.">The endothelial glycocalyx protects against myocardial edema.</a> Circulation Research. 92 (6), 592-594 (2003).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276 (16), 13283-138 (2001).</li><li>Vink, H., Duling, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circulation Research. 79, 581-589 (1996).</li></ol>

我们使用从两层模型和Hertz理论建模的循环血液中的白细胞与血管内皮壁的相互作用的计算出的值。我们计算一个微绒毛上与10 PN负载下的直径为50nm的白细胞会缩进约150nm到糖萼，只有一小部分的总厚度。这表明，糖复合物，与如在本实验中测得的性能，是一种显着的细胞 - 细胞相互作用的障碍，并可以是一个大的空间位阻的细胞期间必须克服的白细胞粘附的粘附级联期间。 ?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>麦考伊的中</td><td> Gibco公司</td><td> 16600-082 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070 </td><td></td></tr><tr><td>血管内皮细胞生长培养基</td><td>血管内皮细胞技术</td><td> MCDB-131 </td><td></td></tr><tr><td>汇集人类脐静脉内皮细胞</td><td>血管内皮细胞技术</td><td> PHUVEC/T-25 </td><td></td></tr><tr><td>硫酸</td><td> JT贝克</td><td> 9681-02 </td><td></td></tr><tr><td>过氧化氢</td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> （3 - 氨基丙基）三乙氧基硅烷</td><td> Aldrich公司</td><td> 440140-100ML </td><td></td></tr><tr><td>异丙醇</td><td> VWR </td><td> BDH8999-4 </td><td></td></tr><tr><td>胰蛋白酶</td><td> Cellgro </td><td> 25-054-C1 </td><td></td></tr><tr><td>汉克的缓冲盐溶液</td><td> Gibco公司</td><td> 14175-095 </td><td></td></tr><tr><td>磺基-NHS-LC-生物素</td><td> Thermo Scientific的</td><td> 21335 </td><td></td></tr><tr><td> Streptavadin珠</td><td>磁珠</td><td> 112.06D </td><td></td></tr><tr><td> MFP-3D原子力显微镜</td><td>庇护研究</td><td></td><td></td></tr><tr><td>无针尖悬臂</td><td>纳米世界</td><td> ARROW-TL1-50 </td><td></td></tr><tr><td>剪影SD </td><td> Quickutz </td><td>剪影-SD </td><td></td></tr><tr><td>硅橡胶</td><td>斯托克韦尔弹性体</td><td> SE50-RS </td><td></td></tr><tr><td> 30毫升注射器</TD&gt; <td>本顿迪金森</td><td> 309650 </td><td></td></tr><tr><td> 18号针</td><td>本顿迪金森</td><td> 305196 </td><td></td></tr><tr><td>拓</td><td> Hospira公司</td><td> 4429-48 </td><td></td></tr><tr><td> 4单向阀</td><td>泰利福</td><td> W21372 </td><td></td></tr><tr><td>男/女港口上限</td><td>史密斯医疗</td><td> MX491B </td><td></td></tr><tr><td>蠕动泵</td><td> Watson-Marlow公司</td><td> 401U / D </td><td></td></tr><tr><td>蠕动管</td><td> Watson-Marlow公司</td><td> 903.0016.016 </td><td></td></tr><tr><td>无菌过滤器</td><td>颇尔生命科学</td><td> 4652 </td><td></td></tr></tbody></table>

quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy

我们理解白细胞捕获期间的白细胞与血管壁的相互作用的限制由内皮表面层的机械性能的一个不完整的理解。它是已知的非均匀分布的白细胞粘附分子相对于表面拓朴图3，该地形限制粘结形成其他的表面9，并且，生理上的接触力（≈5.0 - ：10.0，PN每微绒毛）可以为压缩的微绒毛少三分之一其休息的长度，增加分子的可访问性的相对表面3，7。我们认为，血管内皮细胞的两层结构，相对刚性的单元主体，加上糖萼腔表面6上，一个软保护糖衣。它已被证明，糖复合物可以作为阻挡减少白细胞粘附于内皮表面4。在这份报告中，我们着手解决的内皮细胞表面的变形，以了解血管内皮的机械刚度，可能会影响债券的形成。静态培养中生长的血管内皮细胞不表达一个强大的糖复合物，但在生理流动条件下生长的细胞开始观察体内的糖复合物。内皮细胞体的模量，已使用原子力显微镜（AFM）测定的，是大约5到20千帕5。的糖复合物中的厚度和结构进行了研究，使用电子显微镜8，和已糖复合物的模量的近似使用间接的方法，但就我们所知，没有已经发表在活细胞中的蛋白质复合物的弹性模量的直接测量的报告。在这项研究中，我们提出了一个新的原子力显微镜探针对细胞已培养的条件下，最大限度地发挥他们的糖被表达马压痕实验科的直接测量的弹性模量和厚度的内皮糖萼。

在一个典型的实验中，从一个给定区域的细胞得到的20的力与距离曲线，通常在核周区域，靠近，但不包括在核（〜2μm的范围内）。曲线，考虑到对准测量的持续时间的任何样品漂移，然后取平均值，以除去悬臂噪音，如在图4中示出。曲线进行了分析和合适的被开发用于确定的弹性模量和厚度的薄的聚合物膜1的两个层模型。从拟合的曲线的25个细胞中，我们已经确定，管腔?...

Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

内皮糖萼的机械特性测定压痕使用AFM的悬臂上的微米大小的球体。内皮细胞培养在一个自定义的腔室，在生理流动条件下，诱导糖萼表达。使用薄膜的模型，以确定具有糖萼厚度和弹性模量的数据进行了分析。

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Research

JoVE Journal

Bioengineering

Bacterial Immobilization for Imaging by Atomic Force Microscopy

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

科研

生物工程

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 ...

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, ...

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 ...

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 ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

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 ...