Name: adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface
Uploaded: 2011-11-02T13:00:00
Description: Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

这项研究是由NIH的拨款R01HL091020，R01HL093723，R01AI077343，并R01GM096187支持。

1。从全血红细胞隔离<ol><li>准备EAS - 45解决方案。重达从表一的所有成分，并溶于DI水100 200毫升。加水使千毫升解决方案，并调整pH值至8.0。过滤和分装50毫升。冻结储存在-20 ° C。 </li></ol>注：作为一名护士，应当由受过训练的医疗专业等步骤1.2机构审查委员会批准的协议，。 <ol start="2"><li>绘制成10毫升含有EDTA管中位数肘静脉3血5毫升，轻轻混匀血用EDTA，立即?...

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要成功使用微管粘附频率检测应考虑的几个关键步骤。首先，确保记录的具体利益的受体 - 配体系统的相互作用。非特异性控制测量（参见图3，4）保证的特殊性。理想的情况下，非特异性黏附概率应低于0.05，对所有接触的时间段，每个时间点之间的特异性和非特异性黏附概率有显著差异。不同的方法可以用来夫妇的红细胞表面的配体。结果表明，氯化铬耦合方法17了非特异性结合的生物...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td> 10X PBS </td><td> BioWhittaker </td><td> 17 - 517Q </td><td>用去离子水稀释到1X使用前</td></tr><tr><td> Vacutainer EDTA </td><td>屋宇署</td><td> 366643 </td><td>红细胞隔离</td></tr><tr><td> 10ML PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> Sigma - Aldrich公司</td><td> 10771 </td><td>红细胞隔离</td></tr><tr><td>腺嘌呤</td><td> Sigma - Aldrich公司</td><td> A2786 </td><td> EAS - 45准备</td></tr><tr><td> D -葡萄糖（葡萄糖） </td><td> Sigma - Aldrich公司</td><td> G7528 </td><td> EAS - 45准备</td></tr><tr><td> D -甘露醇</td><td> Sigma - Aldrich公司</td><td> 6360 </td><td> EAS - 45准备</td></tr><tr><td>氯化钠（NaCl） </td><td> Sigma - Aldrich公司</td><td> S7653 </td><td> EAS - 45准备</td></tr><tr><td>磷酸三钠，磷酸氢二铵（娜＆＃X2082; HPO₄） </td><td> Fisher Scientific则</td><td> S374 </td><td> EAS - 45准备</td></tr><tr><td> L -谷氨酰胺</td><td> Sigma - Aldrich公司</td><td> G5763 </td><td> EAS - 45准备</td></tr><tr><td>生物素 - X - NHS </td><td> Calbiochem </td><td> 203188 </td><td>红细胞生物素</td></tr><tr><td>二甲基甲酰胺（DMF） </td><td> Thermo Scientific的</td><td> 20673 </td><td>红细胞生物素</td></tr><tr><td>硼酸盐缓冲液（0.1M） </td><td>电子显微镜科学</td><td> 11455-90 </td><td>红细胞生物素</td></tr><tr><td>链霉亲和素</td><td> Thermo Scientific的</td><td> 21125 </td><td>配体functionalizing </td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A0336 </td><td>配体functionalizing </td></tr><tr><td> Quantibrite体育珠</td><td> BD公司</td><td> 340495 </td><td>密度定量</td></tr><tr><td>流式细胞仪</td><td>屋宇署Immunocytometry系统</td><td> BD LSR II </td><td>密度泉tification </td></tr><tr><td>毛细管 0.7 - 1.0MM × 30“ </td><td>金布尔玻璃公司</td><td> 46485-1 </td><td>微管拉</td></tr><tr><td>矿物油</td><td> Fisher Scientific则</td><td> BP2629 - 1 </td><td>商会大会</td></tr><tr><td>显微镜盖玻片</td><td> Fisher Scientific则</td><td> 12 - 544 - G </td><td>商会大会</td></tr><tr><td>聚乙烯α-人类CD11a 克隆您好111 </td><td> eBioscience公司</td><td> 12-0119-71 </td><td>试剂图</td></tr><tr><td> PE抗人类CD54的</td><td> eBioscience公司</td><td> 12-0549 </td><td>试剂图</td></tr><tr><td>鼠IgG1同型对照PE </td><td> eBioscience公司</td><td> 12-4714 </td><td>试剂图</td></tr><tr><td>液压显微</td><td> Narishige </td><td> MO - 303 </td><td>微管系统</td></tr><tr><td>机械手</td><td>新港</td><td> 461 - XYZ - M，SM - 13，DM - 13 </td><td>微管系统</td></tr><tr><td>压电翻译</td><td>玛格力Instrumente </td><td> P - 840 </td><td>微管系统</td></tr><tr><td> LabVIEW的</td><td>美国国家仪器公司</td><td> 8.6版</td><td>微管系统</td></tr><tr><td> DAQ板</td><td>美国国家仪器公司</td><td> USB - 6008 </td><td>微管系统</td></tr><tr><td>光学平台</td><td>动力学系统</td><td> 5200系列</td><td>微管系统</td></tr></tbody></table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

微管黏附实验是在1998年开发的，衡量的二维（2D）受体-配体结合动力学。检测使用传感器粘附细胞相互作用的分子之一，并提出一个人的红细胞（RBC）的。它采用显微操作与另一个细胞的表达与精确控制的区域和时间，使债券形成的其他相互作用的分子接触，使红细胞。红细胞伸长后发现，除了拉动两个细胞黏附事件。通过控制固定的红细胞表面上的配体的密度，粘连的概率保持在0和1之间的中档。粘连的概率估计在两个细胞之间的一个给定的接触时间的反复接触，周期序列的粘附事件的频率。不同的接触时间，产生一个具有约束力的曲线。拟合概率模型的结合受体-配体反应动力学1曲线返回2D的亲和力和关闭率。 该试剂盒已通过验证，使用与IgG的Fc 段 1-6，选择素与糖缀合物的配体6-9，与配体10-13 homotypical cadherin 的约束力14，T细胞受体与辅助受体肽主要组织相容性复合物15整合Fcγ 受体的相互作用- 19。 该方法已用于生物物理因素，如膜microtopology 5，膜锚2，分子取向和长度为6，承运人刚度9，曲率20，和撞击力20，以及生化因素，量化的二维动力学的规定，如调制的骨架和膜的微环境相互作用的分子， 这些分子表面，15,17,19组织。 该方法也被用来吨Ø研究并发约束力的双种受体-配体3,4，和19三分子相互作用使用修改后的模型 21 。 该方法的主要优点是，它允许在他们的母语膜环境受体的研究。结果可能会非常不同，从获得使用纯化的受体 17 。它还允许在时间分辨率，远远超出了典型的生化方法分一秒的时间尺度的受体 - 配体相互作用的研究。 要说明的微量粘附频率的方法，我们动力学测量间粘附分子-1（ICAM - 1）红细胞约束力的解决方案中的E -选择与二聚体的中性粒细胞激活α大号β2整合素α大号β2功能。

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

粘附频率检测，用于测量当两个分子相互作用的细胞表面锚定受体 - 配体相互作用的动力学描述。使用一个微管加压作为粘附传感器的人体红血细胞和整合αLβ2和细胞间粘附分子-1受体和配体相互作用，这种机械为基础的检测例证。

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

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JoVE Journal

Bioengineering

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Quantitative FRET FÃƒÆ’Ã‚Â¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

一种三肽稳定的油酸纳米乳液

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

分子弹簧常量分析由生物元原力探针光谱

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...