Name: static adhesion assay for the study of integrin activation in t lymphocytes
Uploaded: 2014-06-13T14:00:00
Description: Watch this Scientific Journal Video about Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes at JoVE.com

Hirschil信托，迈克尔·萨珀斯坦医学学者研究基金，以及纽约大学的奖学金白石支持这项工作。

1，涂料的微孔板注意：该步骤的目的是涂层的聚苯乙烯表面与ICAM-1作为配体的T细胞LFA-1。 <ol><li>准备了以下解决方案： <ol><li>通过再悬浮制备涂布液的重组ICAM-1，用磷酸盐缓冲盐水（PBS）溶液（137 mM氯化钠，2.7 mM的氯化钾，10毫米的Na 2 HPO 4，2mM的KH 2 PO 4，pH 7.4）中富含钙（1毫的CaCl 2）和镁（2纳米的MgCl 2）。确保ICAM-1的最终...

<ol>
	<li>Dustin, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+as+messengers+in+T+cell+adhesion+signaling.">Membranes as messengers in T cell adhesion signaling.</a> Nat Immunol. 5 (4), 363-372 (2004).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases+and+LFA-1+reciprocally+modulate+adhesion+and+signaling.">GTPases and LFA-1 reciprocally modulate adhesion and signaling.</a> Immunol Rev. 218, 114-125 (2007).</li><li>Rose, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+modulation+and+signaling+in+leukocyte+adhesion+and+migration.">Integrin modulation and signaling in leukocyte adhesion and migration.</a> Immunol Rev. 218, 126-134 (2007).</li><li>Alon, R., Feigelson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-triggered+leukocyte+arrest:+force-regulated+bi-directional+integrin+activation+in+quantal+adhesive+contacts.">Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts.</a> Curr Opin Cell Biol. 24 (5), 670-676 (2012).</li><li>Griffith, J. W., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cells+in+motion:+migrating+toward+improved+therapies.">Targeting cells in motion: migrating toward improved therapies.</a> Eur J Immunol. 43 (6), 1430-1435 (2013).</li><li>Dustin, M. L. <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+molecules+and+actin+cytoskeleton+at+immune+synapses+and+kinapses.">Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.</a> Curr Opin Cell Biol. 19 (5), 529-533 (2007).</li><li>Springer, T. A., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inside-out+signaling+and+the+immunological+synapse.">Integrin inside-out signaling and the immunological synapse.</a> Curr Opin Cell Biol. 24 (1), 107-115 (2012).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1+regulates+lymphocyte+adhesion+via+upregulation+of+Rap1+at+the+plasma+membrane.">D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.</a> Mol Cell Biol. 29 (12), 3297-3306 (2009).</li><li>Bivona, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rap1+up-regulation+and+activation+on+plasma+membrane+regulates+T+cell+adhesion.">Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion.</a> J Cell Biol. 164 (3), 461-470 (2004).</li><li>Zeltzer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+of+CD4++T+cells+from+dialysis+patients+to+extracellular+matrix+and+its+components+fibronectin+and+laminin.">Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin.</a> Nephrol Dial Transplant. 12 (12), 2618-2622 (1997).</li><li>Bhattacharyya, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+adhesion+to+immobilized+vitronectin+and+Fc+epsilon+RI+cross-linking+cause+enhanced+focal+adhesion+kinase+phosphorylation+in+murine+mast+cells.">Both adhesion to immobilized vitronectin and Fc epsilon RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.</a> Immunology. 98 (3), 357-362 (1999).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li></ol>

有几个实验来研究信号与LFA-1的活化和T细胞粘附12。流式细胞术是用于使用结合选择性要么弯曲或延伸LFA-1单克隆抗体来测量LFA-1的亲和力状态中活细胞。一个这种方法的局限性是，它并没有考虑到整联“亲合力。迁移测定是有用的工具，但他们测量的迁移，而不是微不足道的密合性在特定的时间点。小鼠模型来研究T淋巴细胞的迁移（ 如皮肤过敏模型）是生理有关，但是利用这些模...

T淋巴细胞的粘附是在免疫应答中1的基本处理。这是需要的T细胞相互作用与内皮细胞装潢毛细管壁，对于淋巴结内扫描的抗原呈递细胞（APC），并为免疫突触的形成（IS）与靶细胞2。这些要求在功能上和动力学截然不同。淋巴细胞外渗的过程包括趋化，滚动，粘附牢固，和轮回。滚动坚定的附着力过渡需要T细胞快速响应G蛋白偶联受体信号。这种反应产生，减缓和逮捕的滚动单元3的整联配体的相互作用。在整合的亲和力立即改变介导了这一过程。迁移需要动态相互作用betweenTcells和血管内皮细胞与形成粘连的“前端”和粘连在“后端”破损同的成形和分断4之间大约一分钟的时间间隔。在IS形成inminutes，但需要保持不变为6小时。 有趣的是，1粘附分子，整合素家族成员淋巴细胞功能相关抗原（LFA） - 1，是对所有这些过程5是必不可少的。 LFA-1介导的粘附通过相互作用与免疫球蛋白超家族的几个成员。最广​​泛研究的配体​​与LFA-1的最高亲和力的细胞间粘附分子（ICAM）-1。非活化的外周血淋巴细胞表达低亲和性的LFA-1在细胞表面上，因此不能粘附的ICAM-1包被的表面。 LFA-1的亲和力是可变的，通过几个信号传导事件，例如G蛋白偶联受体活化，细胞因子的刺激，并通过T细胞受体（TCR）介导的信号调节。的LFA-1表达的细胞内活化到细胞外空间将得到的高亲和性的形式吨hrough相互作用与ICAM-1。这个途径被称为由内而外的信令7。同样地，通过LFA-1从细胞外空间的信号被称为由外向内的信令。 在细胞内信号参与由内而外和由外而内的信号级联是当前研究的一大重点。小GTP酶Rap1的最近成为的由内而外的重要组成部分信令是常见的两种TCR结扎和细胞因子信号8。 Rap1的中整合素激活的关键作用是由Rap1的过度表达刺激T细胞的整合素依赖性粘附的发现凸显，而T细胞粘连阻塞由显性负Rap1的9的表达。在我们的整合调控Rap1的理解这些进展利用体外工具已经完成。其中之一是这里所描述的静态粘附实验。 这种方法的总体目标是研究Ŧ细胞粘附性的ICAM-1包被的表面。更具体地讲，它是用来客观地测量和量化向着它的反配体LFA-1的亲和力在活细胞中的实时性，在不同条件下。这种技术使用涂有ICAM-1的模拟蜂窝表面的T细胞相互作用聚苯乙烯孔。许多先前所描述的静态T细胞黏附实验是实验复杂。这些通常需要T细胞的测定法进行放射性标记，用于培养的牛角膜细胞建立的细胞外基质作为基质的T细胞粘附，或称为非生理性T细胞的刺激在延长的持续时间，以促进T细胞粘附10。利用荧光测量以下的粘附培养量化T细胞是一种更敏感和准确的定量的方法相比，流式细胞术和显微术，如被用在许多其他的检测系统11。另外，单电池microscop整本地化的集成电路分析不允许以同样的方式作为荧光测量的宽峰，基于群体分析。而激活态特异性LFA-1抗体是市售的，这些抗体具有低灵敏度相对于此处所述的方法。过替代技术的主要优点是它的简单性，并同时检测多种实验条件的能力。当考虑这个方法，一个特定的应用，应该考虑到，T细胞应阴性选择，新鲜分离的，并用荧光标记物。

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Plate reader</td>
			<td style="width:160px;">BioTek&#160;</td>
			<td style="width:136px;">Synergy H1 Hybrid</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Multichannel pipette</td>
			<td style="width:160px;">Fisher</td>
			<td style="width:136px;">21-377-829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">96-well microplate with optical bottom</td>
			<td style="width:160px;">Corning Costar</td>
			<td style="width:136px;">3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Recombinant ICAM-1</td>
			<td style="width:160px;">R&#38;D Systems</td>
			<td style="width:136px;">ADP4-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Anti-CD3 antibodies</td>
			<td style="width:160px;">Ancell</td>
			<td style="width:136px;">144-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">PMA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">79346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">BSA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">CFSE</td>
			<td style="width:160px;">Molecular Probes</td>
			<td style="width:136px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">RPMI</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">DPBS containing calcium and magnesium</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Peripheral lymphocytes</td>
			<td style="width:160px;">e.g. mouse or human</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Open Gen 5 software</td>
			<td>BioTek</td>
			<td>Version 2.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 and 50 mL centrifuge tubes</td>
			<td>Fisher</td>
			<td>352099, 352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-75 culture flasks</td>
			<td>Fisher</td>
			<td>50-754-1366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RosetteSep T cell enricment kit</td>
			<td>StemCell Technologies</td>
			<td>15061</td>
			<td></td>
		</tr>
	</tbody>
</table>

static adhesion assay for the study of integrin activation in t lymphocytes

T淋巴细胞的黏附是必需的多种T细胞的功能，包括迁移到炎症和形成免疫突触与抗原提呈细胞的部位。 T细胞的调节完成粘附通过控制整，一类细胞粘附分子组成的异源二聚体对与靶分子上的伴侣细胞或细胞外基质相互作用的跨膜蛋白的的粘接性能。最突出的T细胞的整合素是淋巴细胞功能相关抗原（LFA）-1，亚基αL和β2，其目标是在细胞内粘附分子（ICAM）-1组成。 T细胞来控制粘附的能力来自于调节个人整合的亲和力状态的能力。由内而外的信令描述了进程，使细胞内的信号导致整合的外部域假设激活状态。大部分我们对这些复杂现象的知识是基于机械在简化进行体外模型系统研究。这里所描述的T淋巴细胞粘附实验是一个极好的工具，允许T细胞附着到目标分子，在静态条件下，然后利用荧光板读数器对量化的粘合性。该测定法已在界定粘附刺激性或抑制性物质对淋巴细胞作用，以及表征所涉及的信号转导事件是有用的。虽然这里描述为LFA-1 - ICAM-1介导的粘附;此测定法可容易地适于允许的其他粘合剂的相互作用（ 例如，VLA-4 -纤连蛋白）的研究。

下面是使用与各种浓度的抗-CD3抗体的刺激的初级T细胞粘附试验的一个例子。它是有用的知道洗过细胞的荧光（ 即总负荷）。未刺激的细胞作为阴性对照和PMA处理的细胞是阳性对照的抗-CD3抗体。细胞接种于无涂层的水井作为ICAM-1的控制。在一个典型的实验中附着的PMA处理的细胞的百分比在40％至50％，而未受刺激的细胞的百分比在5％至10％。量化细胞粘附性的另一种方法是通过荧光强度的?...

Watch this Scientific Journal Video about Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes at JoVE.com

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

静电粘附实验对整合素激活的T细胞研究

静态粘合试验是一种可以用于建模的T淋巴细胞和其他细胞类型之间的相互作用的有力工具。通过标记的T细胞注入到井中涂有粘附分子，而读板器是用于以下串行洗涤量化贴壁细胞的数量所产生的相互作用。

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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