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的最终浓度为10微克/毫升，从0.7毫克/毫升的储备溶液制作并保存在-80℃冰箱。 </li><li>通过丰富的PBS与0.5％人（或牛）血清白蛋白（HSA / BSA），2毫摩尔MgCl 2，和1mM的CaCl 2制备的粘合溶液。 </li><li>通过加入2毫摩尔MgCl 2和1mM的CaCl 2，以PBS中制备洗涤液。温暖所有的解决方案37°C使用前。 </li></ol></li><li>用50μl的洗涤液中洗涤24个孔。这将包括未洗井，正（PMA处理的细胞）和阴性（无涂层的孔中）控制。对于每个条件设置至少三个井（一式三份）。 </li><li>加50微升涂布液到每个孔中。不要添加未涂布对照孔。孵育内37℃培养箱中培养一小时。 </li><li>抽吸涂覆溶液轻轻地，不使前端触碰到井的底部。用50μl的洗涤液洗一次。 </li><li>加入50微升的粘附性解决方案和孵化微孔板1小时内37℃培养箱中培养。 </li><li>吸用50微升洗涤液轻轻洗一次。使用该板在同一天，但是也可以将其保持在4℃过夜，并用它翌日。 </li></ol> 2，从外周血T细胞分离<ol><li>增加人体T细胞富集的鸡尾酒在50微升/毫升邻楼全血（ 如 3毫升抗凝剂（肝素，EDTA或柠檬酸盐）全血，加入150μl的鸡尾酒）。拌匀。 </li><li>孵育20分钟，在室温下进行。 </li><li>到50ml的离心管中加入3毫升处理过的血液和PBS等体积的（没有钙和镁）富含1％D-葡萄糖在室温下。用巴斯德吸管轻轻混匀。 </li><li>添加15毫升菲可 - 帕克PLUS的离心管中。 </li><li>仔细层6毫升稀释血液样品的淋巴细胞分离介质上。 </li><li>离心400×g离心20分钟，在20℃，折断。 </li><li>吸干使用干净巴斯德吸管，而使淋巴细胞层原状界面处的上层。 血浆的上层可以被保存以供以后使用。使用干净的巴斯德移液管的淋巴细胞层转移到一个干净的离心管中。 </li><li>添加PBS的3倍体积（9毫升）中的淋巴细胞。轻轻悬浮细胞DRA翼他们进出巴斯德吸管的。 </li><li>离心机在400×g离心5分钟，在20℃下</li><li>除去上清液。淋巴细胞现在应该在培养基中适当的应用程序被暂停。转移细胞对T-75培养瓶中，在20毫升含有10％FBS，1％青霉素/链霉素的RPMI 1640培养基中。 </li></ol>该细胞3。下游注意：一个先决条件是对细胞进行标记，用荧光试剂。在这个协议中使用的羧基荧光素琥珀酰亚胺酯（CFSE），但绿色荧光蛋白（GFP）表达的细胞是一种可接受的选择。 <ol><li>使用血球计数2.4×10 6个T细胞（1×10 5，每孔）。 </li><li>悬浮细胞在培养基中缺乏血清（血清饥饿）。孵育在37℃培养箱中培养的细胞2小时。 </li><li>离心细胞5分钟（400×g离心）。吸媒体和重新悬浮于1ml PBS中。 </li><li>加入CFSE在1:1000稀释（股票，以5毫米制造）。从灯罩后，于室温下8分钟。 </li><li>停止反应，加入10ml的37℃的PBS和自旋，在400×g离心5分钟。 </li><li>重悬细胞沉淀1.2毫升预热的密合性溶液（每50μl1×10 5个细胞）。 </li></ol>的细胞4。刺激注：为了启动粘连，细胞必须被刺激。在下面的实验中，细胞通过T细胞受体使用抗-CD3抗体交联刺激，但是可溶的SDF-1可以作为一种替代方法。取决于实验的目的，多种药理试剂可能之前或期间刺激被添加来研究细胞粘附的影响。佛波醇肉豆蔻酸乙酸酯（PMA）是一个佛波醇酯是在结构上类似于所述第二信使二酰基甘油（DAG），因此激活多种蛋白激酶s下行的T细胞受体（主要是蛋白激酶C）;这里它被用作阳性对照。 <ol><li>温暖的微孔板至37°C。 </li><li>分裂细胞进入8个空1.5毫升管（150微升每管），一管为每个条件。 </li><li>相应地处理细胞： <ol><li>刺激一管与物业管理公司在10毫微克/毫升，作为阳性对照。保持三根管治疗，作为对照用于刺激（“无刺激”），未洗涤的负载控制（“不洗涤”），并且控制对ICAM-1的涂层（“未包衣”）。 </li><li>激发四个管子用不同浓度的抗CD3抗体（0.1，1，5，和10微克/毫升）。继续而不延误下一个步骤。 </li></ol></li><li>分装50μL刺激细胞混合到每个空井。每孔细胞的最终数量为1×10 5。 </li><li>将微孔板在37℃培养箱中培养15分钟。 注意：某些T细胞系需要更短的增产<html> tion时间。在这段时间内，细胞将沉淀下来到孔的底部，并且附着到目标配体。 </li></ol> 5，洗去非贴壁细胞注意：该步骤的目的是去除细胞，是不能够形成与配体包被的表面紧密接触。使用以多道移液器此步骤，以确保在相同的物理力施加到所有的孔中。它保持未洗，以协助贴壁细胞的百分比计算所指示的对照孔是非常重要的。 <ol><li>加入150μl的温暖附着力解决方案，每个井。从井中取出介质之前，轻轻摇动板为几秒钟。请勿触摸孔的底部与技巧。 </li><li>重复此步骤三次。吹打进出缓冲时更改吸管的方向。 </li></ol> 6，确定贴壁细胞的百分比_content“&gt;注意：在此步骤中的荧光板读取器用于在每个孔中测量荧光强度的强度与所述贴壁细胞的数目直接相关。 <ol><li>打开平板阅读器。通过点击桌面上的快捷方式图标，打开酶标仪软件。 <ol><li>从“任务”菜单上，单击“新建”来建立一个新的协议。 </li><li>从“操作”菜单中选择“查看”选项。点击“荧光强度”并点击“确定”选项卡。 </li><li>从下拉菜单中选择“激发波长485”和“发射波长528”。点击“确定”选项卡。 </li><li>在“光学”选项菜单中选择“底部”，并点击“确定”选项卡。返回到“操作”菜单，设定温度为37℃，然后单击“确定”。 </li><li>插入到微孔板读板器和单击“运行”。其结果将被导出到excel电子表格。 </li></ol></li><li>计算贴壁细胞用下列公式的百分比： 贴壁细胞的百分比=（读入洗涤井平均荧光强度）/（读入未洗涤的细胞的平均荧光强度）×100％。 </li></ol>

<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>

作者什么都没有透露。

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。

静电粘附实验对整合素激活的T细胞研究

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

static-adhesion-assay-for-study-integrin-activation-t

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JoVE Journal

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Cell Biology

Immunology and Infection

Unit 1

Cell-Matrix Interactions

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15.9K 次观看.  New York University School of Medicine. 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。 

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

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 shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

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 ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...