Name: static adhesion assay for the study of integrin activation in t lymphocytes
Uploaded: 2014-06-13T14:00:00.000+00:00
Duration: 9 min 14 s
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>

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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，但是利用这些模型是多因素的，复杂的执行。这里所描述的静态粘合力测定法的主要优点在于它的测量以简单的方式二者的亲合力和亲和力的能力。这种方法的另一个优点是它能够快速，准确地检测出少量的细胞的能力。当与其它方法相比，它更容易MANIP乌拉特细胞，在功能测定为我们描述一个不同的试剂对待他们。此外，该贴壁细胞用酶标仪，具有手动计数关联消除偏压客观计数。此方法可用于筛选多种药物或基因操作上的附着过程的影响。 但是这种技术是不自由的限制。一个潜在的缺点是，贴壁细胞的百分比是相对数，其可以变化从一个试验到另一个。另一个弱点是，爆炸淋巴细胞不能被使用，因为这些都必须新鲜分离的。此外，随着细胞从冷冻的外周血单核细胞中回收黏附的研究并不一致。重要的是要提的是物业管理公司应该始终是重要的，我们建议在每一个实验中使用它。正和负控制是不成功的故障排除实验的第一步。如遇缺增强粘附在刺激的细胞中，目标配体覆盖的孔中的浓度应进行检查。除了它需要验证，超过95％的细胞是存活，并在壳体的细胞系，它们的生长阶段，应计算。的情况下在相同的条件内显著变异性，建议使用多于三个相同的孔（大于一式三份）。 为了理解该试验是要明白，在协议中的一些步骤如培养时间，接种细胞的数量必须是均匀的所有条件中是有用的。在培养时间的微小差异可能会导致不准确的测量。它也是重要的等分试样刚好细胞相同数量的是每个孔中。这项技术的未来应用，提高其精度将是将加载的细胞，更客观的方式进行洗涤的自动化版本。此外，一个自动化的版本将使我们执行大型屏幕。

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><tbody><tr><td>Plate reader</td><td>BioTek</td><td>Synergy H1 Hybrid</td><td></td></tr><tr><td>Multichannel pipette</td><td>Fisher</td><td>21-377-829</td><td></td></tr><tr><td>96-well microplate with optical bottom</td><td>Corning Costar</td><td>3603</td><td></td></tr><tr><td>Recombinant ICAM-1</td><td>R&amp;amp;D Systems</td><td>ADP4-050</td><td></td></tr><tr><td>Anti-CD3 antibodies</td><td>Ancell</td><td>144-020</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>79346</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A2058</td><td></td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C1157</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>DPBS containing calcium and magnesium</td><td>Gibco</td><td>14190250</td><td></td></tr><tr><td>Peripheral lymphocytes</td><td>&lt;em&gt;e.g.&lt;/em&gt; mouse or human</td><td></td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>499609</td><td></td></tr><tr><td>Open Gen 5 software</td><td>BioTek</td><td>Version 2.01</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>71-7167-00</td><td></td></tr><tr><td>15 and 50 ml centrifuge tubes</td><td>Fisher</td><td>352099, 352070</td><td></td></tr><tr><td>Disposable plastic Pasteur pipettes</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>T-75 culture flasks</td><td>Fisher</td><td>50-754-1366</td><td></td></tr><tr><td>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计算倍的每个条件较未刺激的细胞。 表1示出的CFSE荧光强度标记的初级T细胞刺激了不同剂量的可溶性抗-CD3抗体以及镀上的ICAM-1包被的孔， 图1示出了代表性的即时通讯年龄相似的实验。将细胞负从外周血中选择。血清饥饿2小时后，2.4×10 6个细胞用CFSE标记后跟刺激与可溶性抗CD3抗体以各种浓度。刺激的细胞铺板到ICAM-1包被的孔，并温育在黑暗中15分钟。温育后，非贴壁细胞洗涤三次，并用酶标仪在485nm处的荧光强度进行测定。 PMA处理和未刺激的细胞作为对照。注意，第一柱（图1A-1C）包含未洗涤的细胞（总负荷， 例如 100％）。 图2示出了T细胞粘附计算根据记录在表1中的荧光强度的百分比。对于每一个条件的3孔中（一式三份）的平均强度进行了计算，并转换成相对百分比出装总细胞（ <stroNG&gt;表1;专栏1）。计算出的平均值的标准误差为每个条件。如该图所示，贴壁细胞的百分比大于与用于刺激抗-CD3抗体的浓度较高。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>没有洗</td><td>胶版纸</td><td>未刺激</td><td> PMA </td><td>抗CD3 0.1微克/毫升</td><td>抗CD3 1微克/毫升</td><td>抗CD3 5微克/毫升</td><td>抗CD3 10微克/毫升</td></tr><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td></tr><tr><td>一</td><td> 89652 </td><td> 611 </td><td> 5219 </td><td> 45873 </td><td> 8964 </td><td> 20157 </td><td> 37972 </td><td> 37972 </td></tr><tr><td>乙</td><td> 90248 </td><td> 320 </td><td> 6049 </td><td&gt; 44698 </td><td> 7568 </td><td> 25486 </td><td> 32549 </td><td> 32549 </td></tr><tr><td> Ç </td><td> 88321 </td><td> 409 </td><td> 5456 </td><td> 42697 </td><td> 8542 </td><td> 24568 </td><td> 32892 </td><td> 3289 </td></tr></tbody></table> CFSE的表1中。荧光强度值标记与不同浓度的可溶性抗-CD3抗体的刺激的初级T细胞 。的新鲜收获的细胞进行血清饥饿2小时，然后用CFSE标记在5μM浓度。可溶性的抗CD3抗体接着，细胞用低（0.1微克/毫升），介质（1微克/毫升），高（5微克/毫升），和非常高（10微克/毫升）的浓度和1个×10 5个细胞接种于各孔中（预涂覆有ICAM-1）。 15分钟后，通过三个串行洗涤除去非粘附细胞。贴壁细胞的数量，测定用酶标仪为shown在此表中。井1A-1C:洗过的细胞;图2A-2C:未涂覆的孔中;图3A-3C:未刺激的细胞; 4A-4C:细胞与10 ng / mL的PMA刺激;图5A-5C:细胞与低剂量的抗-CD3抗体刺激;图6A-6C:细胞与抗CD3抗体培养基的剂量刺激;图7A-7C:细胞与高剂量的抗-CD3抗体刺激;图8A-8C:细胞以非常高的剂量的抗-CD3抗体的刺激。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51646/51646fig1highres.jpg" src="/files/ftp_upload/51646/51646fig1.jpg" /> 图1用不同浓度的可溶性抗-CD3抗体的刺激的初级T细胞的图像 。新鲜收获的细胞进行血清饥饿2小时，然后用CFSE标记在5μM浓度。接着，将细胞用PMA（10纳克/毫升）或不同浓度的抗CD3抗体（0.1，1，5，和10微克/毫升），并镀上ICAM-1包被的表面。代表图像是使用20X放大倍率拍摄的700蔡司共聚焦显微镜。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51646/51646fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51646/51646fig2highres.jpg" src="/files/ftp_upload/51646/51646fig2.jpg" /> 图2的T细胞与高剂量的抗-CD3抗体的结果增加了粘附性刺激的T细胞粘附的百分比的计算从表1中所示。算出粘附细胞的平均百分比相对于未洗细胞。作图表示该代表100％。直方图呈现至少3口井的结果（平均值±标准误差）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51646/51646fig2large.jpg" target="_blank">请CLIC</a>ķ这里查看此图的放大版本。

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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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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

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

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

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

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

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 ...

生物工程

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

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

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

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

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 ...

人中性粒细胞中 β2 整合素激活抑制剂的筛选

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

一种基于流式细胞术的高通量技术筛选整合素抑制药物

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to&#160;focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of&#160;the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading.

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate ...

静电粘附实验对整合素激活的T细胞研究

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此视频中的章节

迁移人T淋巴细胞分离，培养和分析

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

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

用于量化生长因子介导的整合素力学和附着力的张力计系绳探头