Name: assay of adhesion under shear stress for the study of t lymphocyte-adhesion molecule interactions
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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的介质。 </li><li>算使用血细胞计数器的CHO-ICAM细胞，并计算每室0.75×10 5个细胞。注意:在这个实验:将需要2.25×10 5个细胞播种3的腔室。 </li><li>离心机0.75×10 5个每室的CHO-ICAM细胞5分钟，在500 xg离心，室温，并重新悬浮在每CHO的室30微升细胞沉淀-ICAM完整的培养基。 注:在这个实验中，将需要90微升种子3室。 </li><li>慢慢加入细胞进入流室的一个储存器。使细胞在37℃培养箱，用5％ 的 CO 2沉降5分钟。 </li><li>加入200μl完全培养基穿过每个腔室的两个储存器。两者之间交替加法，以避免细胞的运动作为系统达到平衡。 </li><li>孵育细胞过夜37℃培养箱，用5％ 的 CO 2的内部。 </li></ol> 2. T细胞的制备注意:此测定法可以与初级人T细胞或T细胞系​​进行。从血T细胞分离的协议已如前所述。10如果使用原代人T细胞用新鲜分离细胞，以取得最佳效果。如果使用T细胞系，按照由单元的源所指定的培养协议。为了检测在荧光microsco细胞PE的T细胞标记有羧基荧光素琥珀酰亚胺酯（CFSE）。 <ol><li>算使用血细胞计数器的T细胞，并计算每腔室2×10 6个细胞。注意:在这个实验中，6×10 6个细胞将需要3的腔室。 </li><li>离心机2×10 6个 T细胞每室，用于在500 xg离心，室温下5分钟，重悬在1ml磷酸盐缓冲盐水含有1％葡萄糖或2％FBS的细胞（PBS）中。 </li><li> 1添加CFSE:1000稀释液（股票5毫米制造）。从灯盖和在室温下孵育8分钟</li><li>旋转，在500×g离心5分钟，室温。 </li><li>每240条件微升悬浮细胞沉淀（见第4节"说明"）RPMI媒体缺乏血清。在该实验中，重悬细胞沉淀在720微升纯的RPMI。分裂细胞转化为标示每室，每管240微升空1.5毫升管。保存在37℃加热块电池备用。 </li></ol> 3.设置日Ë 输入/输出软管和注射泵<ol><li>暖显微镜实时成像室至37℃。 </li><li>准备输入软管: <ol><li>填的500毫升玻璃瓶与水和使2个孔到盖子。标签上的孔"中"和"out"。这将作为一个加热水浴输入媒体。 </li><li>运行输入通过在盖和进入水浴中的孔冲水。确保连接到注射器的软管的一侧来通过"中的"孔和将连接到输入储存器的一侧来通过"去"的孔。 </li><li>刷新输入用60ml水冲水以从体系中除去任何空气。 </li><li>素输入用RPMI培养基缺少血清冲水加热到37℃。填充60毫升注射器，并通过媒体推，直到软管完全底漆，缺乏任何气泡。 注:根据lengt油管h的使用要求总理将改变音量。 <ol><li>由分钟（5分钟）通过的室的数目的数目在实验（这里3）流量（这里0.3ml /分钟）的速率乘以计算的实验所需的介质的体积;在这个实验中，使用4.5毫升介质。注意:我们建议有5毫升额外的体积（这里共9.5ml）中剩余的注射器吸了。 </li></ol></li><li>将整个输入设置到显微镜的实时成像外壳保持37℃。运行软管和注射器滑出机壳和注射器加载到注射器泵。 </li></ol></li><li>准备输出软管: <ol><li>使在250毫升瓶作为输出废物用的盖的孔。 </li><li>运行通过孔并进入瓶的输出管道。松散的固定盖板，不关闭所有的方式。 </li><li>放置输出设置成英里的实时成像外壳croscope。 </li></ol></li><li>计算的流量（毫升/分钟）必要以产生期望的剪切应力（达因/厘米2）。剪应力在不同血管隔室而变化，因此，考虑到包括待研究的细胞类型并在剪应力水平决定何时治疗的总体模型。 注意:这些实验是在0.4达因/厘米2进行密切模仿小静脉设置。 <ol><li>计算剪切应力时拿腔尺寸考虑在内。使用下面的公式为此处使用的流动室（由生产商11提供）（参见材料表）T =η* 176.1 *直径:其中T =剪切应力η=动态粘度，直径=流速。注意:RPMI培养基在37℃下的动力粘度为0.007。 </li></ol></li></ol> 4.装载细胞的流动室和刺激注意:为了启动粘附，T细胞需要被刺激。在以下实验中，细胞将或者保持未刺激或SDF-1α12或佛波醇肉豆蔻乙酸酯（PMA;阳性对照）被刺激13。对T细胞的趋化因子的适当呈现，CHO-ICAM细胞预温育与SDF-1α（后跟串行洗涤），允许在细胞表面上呈现。 PMA是一个佛波酯在结构上类似于第二信使二酰基甘油（DAG）;此处它被用作阳性对照。 T细胞被直接用PMA装载到流动室前处理。 <ol><li>放置在显微镜流室的幻灯片，并设置使用20X客观的CHO-ICAM单层焦平面。 </li><li>保持CFSE染色T细胞在37℃加热块的所有其他条件。制备的CHO-ICAM或T细胞如下之前立即测定该条件: <ol><li>对于未刺激条件（室1），保持一管/流量通道琥珀未经处理的，以服务为刺激作为阴性对照。 </li><li>对于PMA刺激（室2），刺激T细胞用PMA（10毫微克/毫升），作为阳性对照一管;立即装入流室之前对待。 </li><li>为SDF-1α刺激，（室3），通过每次从上部储存器（ 输出水库）除去70微升3次，然后加入70微升的SDF的刺激与SDF-1α（100毫微克/毫升）的第三流动腔室-1α（100毫微克/毫升）到下部容器（ 输入容器）。孵育5分钟。 </li></ol></li><li>从一个腔室的输出贮存器中删除，并在一个时间丢弃70微升3倍。 </li><li>加70微升预处理（如适用）的T细胞悬液到输入储存器3次。等待5分钟，使细胞从室（"以"）的入口移动，并且到达腔室的近端出口（"输出"）。 </li><li>在此期间，图像5对于CFSE染色T细胞领域。选择分散在整个腔室（ 图1）的中心领域。使用下面的激发/发射条件:激发激光波长:488纳米;排放收集过滤器:500 - 540纳米。这些图像将作为预流动细胞计数。 </li><li>同时附加的输入和输出管连接到流动室水库。 注意:同时附加管路是至关重要的，以便不引入气泡进入室和保持室中整体平衡。 </li><li>以0.3毫升/分钟（0.4达因/厘米2）开始流动，同时可视化的CFSE染色的T细胞。如流程开始，特别是与第一腔室，经常会有引发流动和观察T细胞轧制之间的滞后，因此，在T细胞的运动的发生开始定时5分钟。 </li><li>终止流动5分钟后，并在CFSE通道图像5字段。选择随机分散的救援人员到场领域ughout流动室的中心（ 图1）。这些图像将作为流动后细胞计数。 </li></ol> 5.确定粘附细胞的百分比注意:在所获取的图像中的细胞的数目的测定用自动软件客观完成。对于我们的研究，我们使用的软件Volocity（版本6.2.1），但是其他的软件如ImageJ的（版本1.48V）也是适用的。 <ol><li>确定每个场的预流对于每个条件的细胞的数量。 5场的平均是"流预平均细胞数。"7 </li><li>确定每场的后流的每个条件的细胞的数量。 5场的平均是"流后平均细胞数。"7 </li><li>计算使用下面的公式贴壁细胞的百分比: 贴壁细胞=（流量后平均细胞数）/（流预平均细胞数）的百分比×100％。 </li></ol>

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The authors have nothing to disclose.

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

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 ...

assay-adhesion-under-shear-stress-for-study-t-lymphocyte-adhesion

Research

JoVE Journal

Immunology and Infection

8.4K 次观看.  New York University School of Medicine. 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。 

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

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 ...

科研

生物工程

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 ...

免疫与感染

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

生物化学

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 ...

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

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

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 ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

生物学

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

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

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

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

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