Name: a uniform shear assay for human platelet and cell surface receptors via cone-plate viscometry
Uploaded: 2019-06-05T14:30:00
Description: Watch this Scientific Journal Video about A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry at JoVE.com

与这项研究有关的工作得到了国家卫生研究院 (NIH) 国家心脏、肺和血液研究所的部分支持, 这些研究授予 HL082808、HL123984 (r. l.) 和 F31HL134241 (m. e. q.)。国家卫生研究院国家普通医学研究所也提供了 T32GM008367 (m. e. q.) 赠款;亚特兰大儿童保健和埃默里大学儿童流式细胞术核心的试点拨款。作者感谢 Hans Deckmyn 博士分享6B4 抗体, 并感谢埃默里·儿童儿童研究中心流式细胞术核心提供技术支持。

本文所述的所有使用捐助者衍生的人体血小板的方法都得到了亚特兰大埃默国大学/儿童保健机构审查委员会的批准。
1. 抽血和血小板分离
<ol><li>在实验当天, 通过静脉穿刺将人血从健康的成人献血者身上抽入3.8% 的柠檬酸三钠。一个4.5 毫升的血液管是足够的血小板丰富血浆 (PRP) 为20-25 条件的捐献者, 血小板计数接近 250 x10 3 每μl。 注: 避免通过窄规格针 (小于 21 G)...

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本手稿中描述的协议允许快速和多才多艺的评估层剪切对血小板和细胞表面受体的影响。这里介绍的具体代表性结果强调了剪切流如何影响多价或二价配体的影响。除了这种应用外, 均匀的剪切分析在观察剪切依赖性效应方面也有广泛的应用。在没有已知的配体受体对的情况下, 均匀的剪切分析还可以检测剪切对细胞形状和信号等因素的影响, 尤其是与流式细胞仪分析配对时。图 5 b 部分提到<strong ...

机械感受器是对机械刺激有反应的蛋白质, 如压力或机械摄动/变形。对于一些机械感受器来说, 感知这些机械扰动与它们表达的细胞类型的函数是明确的。以压力感受器神经元中的拉伸受体为例;这些机械敏感离子通道通过感知血管 "拉伸"1,2来调节血压。在内耳, 毛细胞上的离子通道检测声波3引起的机械变形,皮肤低阈值机械感受器 (ltmr) 有助于触觉信息的传递4。在其他情况下, 机械感受器为细胞建立粘附或生长提供了重要信息。细胞可以感觉到其局部环境的刚性, 并可能依靠收缩力通过肌动蛋白细胞骨架和整合素来决定生长或传播5,6。
在研究细胞或组织模型中的受体配体相互作用时, 存在着常见的检测方法, 可以快速、准确地报告温度、ph 值、配体浓度、色调、膜电位和许多其他参数的变化效果它在体内可能会有所不同。然而, 当涉及到检测机械力对受体激活的贡献时, 这些相同的检测方法可能会失败。无论细胞是感知它们的微环境、检测声波还是对拉伸的反应, 上述机械感受器的一个共同点是, 它们参与的相互作用是配体、受体或两者都锚定在表面。为测试机械力对受体相互作用的影响而开发的检测往往反映了这一范式。微流控和流室用于研究剪切流对细胞和受体的影响 7,8。这些类型的实验的优点是允许通过既定的流速微调剪切速率。其他技术使用荧光分子探针来检测细胞在富含配体表面施加的力, 从而准确地读出相互作用 9,10所涉及的力的大小和方向.
除了一个或两个伙伴锚定在表面的情况下发生的机械被组织外, 剪切应力还可能影响溶液中的蛋白质和细胞。这通常是在血液细胞/蛋白质中观察到的, 这些蛋白质在循环中不断存在, 并可能通过激活通常是表面锚定的11 的机械感受器或通过暴露将被阻断的目标序列而表现出来。静态条件12。然而, 测定剪切力对溶液中颗粒的影响的技术相对较少。一些溶液中的方法引入了不同速度和持续时间的流体悬浮液中通过涡旋细胞的剪切, 尽管这些方法可能无法非常精确地确定产生的剪切应力。旋转粘度计通过对流体施加特定的剪切力来测量粘度。本文介绍了一种用于确定特定层流剪切速率对溶液中细胞或细胞碎片的影响的方法。
血小板表面表达最强烈的蛋白质之一是糖蛋白 (GP) Ib-IX 复合物。GPIb-IX 是血浆蛋白 von Willebrand 因子 (VWF) 的主要受体。总之, 这种受体配体对一直被认为是血小板对剪切应力13 反应的基础。在血管损伤的情况下, VWF 与内皮下基质14中暴露的胶原蛋白结合, 从而通过 Vwf-gubib-ix 相互作用将血小板吸收到损伤部位。如果 GPIb-IX 在生理剪切应力作用下引起膜近端机械感域 (MSD) 的展开, 而膜近端机械传感器域 (MSD) 又会激活 Gpiib-ix 15, 则 VWF 与 gpibα亚基中的结合位点接触。在最近的一项研究中, 我们已经表明, 针对 GPIbα的抗体, 就像许多免疫血小板减少症 (ITP) 患者产生的抗体一样, 也能够通过在剪切应力下展开的 msd 诱导血小板信号。然而, 与 VWF 不同的是, 这种抗物质通过在正常循环下固定复合物来促进剪切诱导的 GPIb-IX 激活, 它能够通过 GPIb-IX 交联血小板, 并在循环中展开 MSD。这样, 通常通过剪切作用下的表面固定激活的机械感受器就可以在溶液中激活。在本报告中, 我们将演示如何利用基于粘度的均匀剪切分析来检测特定水平的剪切应力对溶液中受体激活的影响。

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			<td height="34" style="height:35px;width:413px;">APC anti-human CD62P (P-Selectin)</td>
			<td style="width:128px;">BioLegend</td>
			<td style="width:135px;">304910</td>
			<td style="width:88px;"></td>
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			<td height="22" style="height:22px;width:413px;">Brookfield Cap 2000+ Viscometer</td>
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			<td style="width:135px;">-</td>
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			<td height="21" style="height:21px;width:413px;">FITC-conjugated Erythrina cristagalli lectin (ECL)</td>
			<td style="width:128px;">Vector Labs</td>
			<td style="width:135px;">FL-1141</td>
			<td></td>
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			<td height="21" style="height:21px;">Pooled Normal Human Plasma</td>
			<td>Precision Biologic</td>
			<td>CCN-10</td>
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			<td height="52" style="height:52px;width:413px;">Vacutainer Light Blue Blood Collection Tube (Sodium Citrate)</td>
			<td style="width:128px;">BD</td>
			<td style="width:135px;">369714</td>
			<td></td>
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			<td height="53" style="height:53px;width:413px;">Vacutainer Blood Collection Set, 21G x &#190;&#34; Needle</td>
			<td style="width:128px;">BD</td>
			<td style="width:135px;">367287</td>
			<td></td>
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a uniform shear assay for human platelet and cell surface receptors via cone-plate viscometry

许多生物细胞组织通过机械感受器感知其局部环境的机械特性, 这些蛋白质可以对压力或机械扰动等力作出反应。机械感受器通过多种机制检测其刺激并传输信号。机械受体的一些最常见的角色是神经元的反应, 如触摸和疼痛, 或在平衡和听觉中发挥作用的毛细胞。对于经常暴露在剪切应力下的细胞类型, 如内皮细胞、血管线或在正常循环中经历剪切的血细胞, 机械培养也很重要。粘度计是检测流体粘度的设备。旋转粘度计也可用于对流体施加已知的剪切力。这些仪器在流体中引入均匀剪切的能力已被用来研究许多生物流体, 包括血液和血浆。粘度计也可用于将剪切作用于溶液中的细胞, 并测试剪切对特定配体受体对的影响。在这里, 我们利用面板粘度计测试内源性剪切应力水平对血小板的内源性水平的影响, 抗体对血小板机械感觉受体复合物 Gplib-ix 进行治疗。

图 1概述了最初引入的 GPIb-IX 激活触发模型, 以解释锚定血管壁时与熊相关的受体激活, 也可能支持通过多价配体交联的血小板激活。图 2显示了由两种抗体对 GPIb-IX n 端域 (6B4 和 11a8) 和一种在剪切和静态条件下控制抗体 (正常 igg) 进行治疗的人血小板活化读数。在图 3中, 血小板用6B4 和抗体进行了治疗, 其不结合力太低, 无法触发 Gu...

Watch this Scientific Journal Video about A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry at JoVE.com

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

我们描述了一种用邻面色谱法将均匀剪切应用于血小板表面受体的溶液法。这种方法也可以更广泛地应用剪切其他细胞类型和细胞片段, 不需要针对特定的配体受体对。

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

该协议使我们能够回答有关剪切作用于溶液中细胞的多种问题，并显著控制剪切持续时间和幅度。与其他技术不同，这种技术可以检测剪切对固定细胞的影响，它允许我们将剪切应用于溶液中的细胞，并适合一系列下游信号检测方法。在从同意的、健康的成人供体获得外周血液后，在4.5毫升的管中，用3.8%的三钠柠檬酸盐，将血液离心以获得富含血小板的血浆，并使用移液器尖端切入45度角，小心去除由此产生的顶部、多云、黄色血小板丰富的血浆层。通过完整的血液计数获得血小板计数，将血小板计数调整为每微升第五个血小板的2.5倍至第五个血小板，并汇集人类血小板贫乏血浆。然后，将悬浮液在 22 摄氏度下轻轻旋转。对于配体治疗，缓慢地将感兴趣的抗体或配体添加到富含血小板的血浆中，然后轻轻地混合血小板。在孵育过程中，打开圆锥板粘度计，将板温设置为 22 摄氏度。5 到 10 分钟后，将经过处理的富含血小板的等离子体直接转移到板中心温度控制的锥板粘度计上，注意所有样品都沉积在接触点锥体和圆锥体之间。要对样品应用剪切，请根据粘度计手册计算剪切，然后以适当的速率和持续时间对样品应用剪切。在应用结束时，将圆锥体从板上抬起约两毫米，使样品与板和圆锥体保持接触，并使用凝胶加载移液器尖端从样品体积中心收集 5 到 10 微升。然后在室温下用抗体对表面标记物孵育20分钟，并在室温下将样品在2%半甲醛中固定20分钟。要通过流式细胞学分析样本，请根据每个荧光因子的强度测量至少 20，000 个事件，并量化每个荧光标记的信号强度。然后使用牛血清白蛋白或车辆的负面对照绘制闸门，排除负背景荧光事件，并量化所有实验条件下门内事件的百分比。用于响应剪切力应用的血小板激活标记表达式在时间依赖性方面不受管制。在这里，在两个针对糖蛋白1b-IX的N-终端域的抗体和一个在剪切和静态条件下对照抗体的两种抗体存在的情况下，具有代表性的人类血小板活化读数。与静态条件或控制抗体存在相比，所有三种激活标记在剪切力和靶向抗体的存在中显著增加。值得注意的是，使用未结合力过低以触发糖蛋白 1b-IX 活化的抗体治疗不会触发在静态或剪切力条件下的血小板激活标记调节。此外，来自免疫血小板减少症患者的血浆含有对糖蛋白1b-IX的抗体触发剪切依赖反应，与来自没有糖蛋白1b-IX靶向抗体的患者的血浆相反。除了流细胞计量分析外，接触过剪切力的细胞也可以被lys化为西印、ELISA和质谱分析，以确定蛋白质水平的任何变化。与一如既往，在使用血液或人体样本时，请务必穿戴适当的防护设备，并遵循您机构的实验室安全和血液出生病原体指南。

Research

JoVE Journal

Biology

A Chromatin Assay for Human Brain Tissue

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

科研

生物学

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

A &#946;-glucuronidase GUS Based Cell Death Assay

一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Glycopeptide Capture for Cell Surface Proteomics

糖肽捕获的细胞表面蛋白组学

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...