Name: assay of adhesion under shear stress for the study of t lymphocyte-adhesion molecule interactions
Uploaded: 2016-06-29T16:00:00
Description: Watch this Scientific Journal Video about Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions at JoVE.com

The Rheumatology Research Foundation and the Hirschil Trust supported this work.

1.电镀CHO-ICAM细胞注意：该步骤的目的是与产生一个汇合单层的目标镀CHO-ICAM细胞在流动室生长过夜。 <ol><li>保持于10cm组织培养处理过的培养皿的CHO-ICAM细胞中，在37℃在补充有10％胎牛血清（FBS）和1％青霉素/链霉素（CHO-ICAM完全培养基），用5％10毫升RPMI培养基的 CO 2。 </li><li>以收集细胞，加入1ml的0.5％胰蛋白酶-EDTA，并在室温下孵育1分钟，轻轻摇动。中和胰蛋白酶用4ml的?...

<ol>
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为了适当地分析T细胞的粘附，将包括在该研究中的兴奋剂选择的体外方法时必须加以考虑。虽然有多种测定法来研究信号导致LFA-1的活化和ICAM-1的结合的方法都不能互换。静态粘附实验10是最适合于研究T细胞APC相互作用;另外，这里详细的剪切应力的方法是理想的模型T细胞上皮细胞间的相互作用在体内 ，趋化因子沿内皮提出，滚动T细胞必须牢牢坚持和抗血液的剪切应力流<sup...

T淋巴细胞的粘附介导许多不同的过程中健康的免疫系统，3中的T细胞贩卖和抗原呈递发挥重要作用。是否免疫监视或主动免疫应答用于粘附这两大作用是至关重要的过程。4的T细胞-内皮细胞相互作用的生理信号事件是从T细胞的抗原呈递细胞（APC）的相互作用不同，因此需要的不同的方法研究以更好地了解所涉及的信号通路。 T细胞向淋巴细胞外渗期间的血管壁的牢固粘附，需要快速和动态的整合激活。沿内皮激活状态整合和粘附分子之间的紧密相互作用导致密合性的血液流动性，从 ​​而使T细胞沿着搜索容许到孔通道区域的表面爬行。5 T细胞双向的抓取-directionally ALONガ血管壁是在偏振粘附依赖，与T细胞的不同粘合剂前端6最重要的，牢固粘附和轮回需要由循环血液流动产生的剪切力的阻力。 当设计实验来研究细胞粘连，应注意感兴趣的特定的刺激。而整合激活是各种形式的T细胞粘附的常见和重要组成部分，活化的级联很可能是唯一的个人受体和共同受体的下游。同样，整合和黏附分子对在专门的微环境和特定亚群的功能。以这种方式，这些对可以相当不同的管制。这里提出的模型是理想的信号级联，导致整活化发生的剪切应力条件下的研究。7这些相互作用不能充分在静态adhesi理解系统由于冲击这些力已显示直接对T细胞的行为。8虽然与T细胞和CHO（中国仓鼠卵巢）细胞工程化以表达人ICAM-1（CHO-ICAM细胞）的系统可以在这里提出很容易被修改，以研究不同白细胞群或粘附分子。 此法提供了使用剪切应力，为白细胞外渗的牢固粘附阶段的模型来量化T细胞的粘附性和整合激活的方法。通过使用CHO-ICAM细胞LFA-1为它的活细胞中配体的亲合性可以实时响应于感兴趣的各种刺激进行检查。需要容易地获得该技术中，在组合市售微流腔室用注射器泵，极大地简化必要的血液流动和剪切应力建模设备相比其他模型9该测定的另一主要优点在于，特定的信令级联和单个整合的所得活化状态可以通过使用表达所关注的人的粘附分子工程改造的CHO细胞的被干净了研究。此外，量化数据的与活细胞成像的组合是此方法的一个显著优势。总体而言，虽然许多静态的T细胞粘附试验中已经描述了很好地建模T细胞的APC的相互作用，这些模型中捕获的T细胞上皮粘附的动态过程不充分。由于这个原因，选择一个粘附试验时有问题的刺激必须加以考虑。

<table border="0" cellpadding="0" cellspacing="0" width="950">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">T cell samples (cell line or primary)</td>
			<td style="width:269px;">ATCC</td>
			<td style="width:119px;">TIB-152</td>
			<td style="width:248px;">Peripheral human T cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHO-ICAM-1 cells</td>
			<td>ATCC</td>
			<td style="width:119px;">CRL-2093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#181;-Slide VI 0.4 ibiTreat</td>
			<td>ibidi</td>
			<td style="width:119px;">80606</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">500 ml glass bottle</td>
			<td>Fisher</td>
			<td style="width:119px;">FB800500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 ml glass bottle</td>
			<td>Fisher</td>
			<td style="width:119px;">FB800250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone tubing 0.8 mm</td>
			<td>ibidi</td>
			<td style="width:119px;">10841</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope with incubator chamber</td>
			<td>Ziess</td>
			<td style="width:119px;">700</td>
			<td>Any wide field fluorescent microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td style="width:119px;">NE-300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 ml syringe</td>
			<td>BD</td>
			<td style="width:119px;">309653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFSE</td>
			<td>eBioscience</td>
			<td style="width:119px;">65-0850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>R&#38;D</td>
			<td style="width:119px;">350-NS-010/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI</td>
			<td>Lonza</td>
			<td style="width:119px;">12-702F/12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS&#160;</td>
			<td>Lonza</td>
			<td style="width:119px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Eppendorf&#160;</td>
			<td style="width:119px;">5424</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMA</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">16561-29-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Volocity software</td>
			<td style="width:269px;">Perkin Elmer</td>
			<td style="width:119px;">Version 6.2.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software</td>
			<td style="width:269px;">NIH</td>
			<td style="width:119px;">Version 1.48V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-culture treated culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.25%) Phenol Red</td>
			<td>Gibco</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat Inactivated FBS</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>BP295950</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay of adhesion under shear stress for the study of t lymphocyte-adhesion molecule interactions

总体而言，T细胞的粘附是功能的关键组成部分，有助于细胞招募的不同的过程，以炎症和与免疫突触的形成抗原呈递细胞（APC）的相互作用的位点。这两个T细胞粘附的背景下在T细胞相互作用的APC可以认为是静态的不同，而T细胞的血管相互作用由循环本身产生的剪切应力的挑战。 T细胞的APC的相互作用被归类为在静态的两个蜂窝伙伴是相对于彼此静止。一般，这种相互作用淋巴结内发生。作为T细胞与血管壁相互作用，细胞逮捕和必须抵制产生的剪切应力。1,2这些差异突出了需要更好地了解流动条件下的静态强度和附着力为两个不同的监管程序。 T细胞粘附的调节可以最简洁地描述为CON曳整合在细胞表面上表达的分子，并从而调节与相互作用细胞的表面上表达的粘附分子的配体整合素相互作用的亲和力状态。我们目前的整合亲和力国家调控的认识来自于体外模型系统常常简单化。使用此处描述的流动条件附着力的测定允许可视化和实时以下一个刺激的T细胞的上皮细胞的相互作用准确定量。流测定下的粘合可以应用于在用抑制或刺激物质治疗的T细胞内信令粘附研究。另外，该测定可超出T细胞信号被扩展到任何粘合剂白细胞种群和任何整合素的粘附分子对。

代表性的结果从使用Jurkat和初级人CD3 + T细胞的流动粘附实验中所示，所指示的，与SDF-1α刺激。在所有所示实验的阴性对照是未刺激细胞。未刺激细胞的基础门槛百分之附着力5之间 - 10％;基附着力特别是高于此范围表示一个有问题的实验，并建议T细胞的起始群体中非特异地在制备预活化。折叠在贴壁细胞中超过未刺激细胞的每个条件流动后可以除了百分之粘附的...

Watch this Scientific Journal Video about Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions at JoVE.com

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

这种流动粘附实验提供了T细胞的上皮细胞间的相互作用一个简单的，高冲击的模型。的注射器泵用于产生剪切应力，和共聚焦显微镜捕捉图像进行定量。这些研究的目标是使用流动条件，有效地量化的T细胞粘附。

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Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

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Introducing Shear Stress in the Study of Bacterial Adhesion

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During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...