作者想感谢爱米丽Mairey和灵光Donnadieu程序的初始设置。

1。人类宿主细胞和细菌培养<ol><li>文化脐静脉内皮细胞之间的通道1和5％（V / V）的CO 2培养箱在37 ° 9。通道的细胞，他们的做法与胰蛋白酶/ EDTA 80％汇合时提供75厘米的培养瓶和一次性流室（μ-幻灯片六）实验文化维护文化。 </li><li>退出要传代培养瓶的培养基，用10毫升的PBS洗细胞，撤回和更换1.5毫升的胰蛋白酶/ EDTA。允许细胞在细胞培养孵化器分离5分钟在37 ° C。 </li><li>收集细胞培养液10毫升到15毫升收集管的细胞。一个5分钟的离心沉淀细胞（200克），在室温下。暂停4毫升内SFM辅以10％的细胞（V / V）FBS和计数上Malassez腔，根据制造商的指示。 </li><li>引入通道（3X10共4个 ）30一个1 × 10 6 HUVEC细胞每毫升悬浮液，并允许坚持3个小时的细胞在培养箱在37 ° 5％（V / V）的CO 2的气氛。添加一个额外体积为120μL内SFM填补了井。细胞应形成subconfluent的单层（图2）。 </li><li>成长N。脑膜炎表达GCB琼脂含有凯洛格的补充和5在37微克/毫升的氯霉素°在一个潮湿的含有5％的气氛彗星（V /板绿色荧光蛋白（在这种情况下的应变8013表达一个IPTG的诱导启动子控制下的GFP）， V）CO 2为16小时。 </li></ol> 2。个别细菌的初始粘附到宿主细胞<ol><li>调整预热远藤- SFM包含120分钟的10％（V / V）FBS和孵化在37 °在潮湿的大气中含有5％（V /彗星OD600值0.05 GCB琼脂平板上生长的细菌含量V）CO 2，下轻轻摇动（130转）。整个孵化期的培养基中加入1 mM的IPTG的诱导GFP的表达。 </li><li>放在倒置显微镜配备了激烈的平台，样品的温度保持在37 ° C。阶段一次性μ- SLIDE </li><li>无菌玻璃烧杯倒入预热远藤- SFM辅以2％（V / V）胎牛血清和填充50毫升无菌注射器，这种媒介。将"进入"管注射器和填写引入介质。 </li><li>连接"入口"的μ- SLIDE油管，小心，以避免空气进入墓室。然后，贴上"退出"管的另一端，并认真填写渠道中"退出"室约1厘米的距离。 </li><li>测量细菌的OD600值和内SFM含有2％（V / V）FBS的调整到0.15。为了寻找个别细菌必须被打乱，任何聚集的细菌样本的蓬勃震荡。 </li><li>广场100μL内SFM进了水库，并辅以2％（V / V）FBS的培养基，添加100μL细菌的解决方案采取的振荡的解决方案，以避免采样任何剩余的细菌总量的顶部。 </li><li>小心地转动活塞腔注入细菌引入到μ-幻灯片200μL体积。 </li><li>引进内SFM含2％（V / V）FBS的，保持在37 °加热平台，进入会议厅使用注射泵剪应力兼容0.044达因/厘米2 15分钟的附着力。 </li></ol> 例如，请参阅视频1或图3。这一步后，可以移动到第3，4或6。 3。个别细菌的初始粘附宿主细胞的定量<ol><li>经过15分钟的初始粘附，十个领域获得的图像随机，而液体至今仍流传。 </li><li>分析获得的图像，使用ImageJ 软件 8，以访问adherant细菌每场平均人数。这样做如下:首先，每个图像使用的"门槛"窗口，在"图像"菜单中找到，同时最大限度地从细胞中的背景，以突出个人坚持的荧光细菌阈值。然后，每一个单一的细菌也计算在内，通过使用插件"细胞计数" <a href="http://rsb.info.nih.gov/ij/plugins/cell-counter.html">（</a> http://rsb.info.nih.gov/ij/plugins/cell-counter.html） 。每幅图像的数据报告在Excel电子表格，并平均量化附着细菌每场平均人数。 </li></ol> 4。测量电阻，个别细菌附着在宿主细胞流<ol><li>计划注射泵设置产生剪应力，从3到100达因/ 厘米 2 5分钟。 </li><li>在5分钟结束时，获得随机选择步骤3.1中所述的10个领域，但与流动停止PRIOR到收购。 </li><li>步骤3.2中所述的分析获得的图像和量化平均每场剩余的细菌数量。 </li></ol> 5。测量一个孤立的附着细菌的生长到microcolony <ol><li>引进内SFM含10％（V / V）胎牛血清，保持在37 ° C，加热平台进入会议厅使用注射泵与所选择的几个小时的剪应力。为每5分钟一帧的实时视频显微镜图像记录。 </li><li>下面的视频显微镜，停止关闭注射泵的剪应力，获得2-3额外的图像，然后停止该软件的图像采集序列。细菌繁殖的一个例子可以看出，在视频2。 </li></ol> 6。测量电阻附着细菌microcolonies的流动<ol><li> （step5.2）收购后的细胞表面形成大microcolonies流应用程序的前2-3图像的两种细胞（相位相反）和细菌（GFP荧光）。其余实验，收购协议将只监视的荧光。 </li><li>高剪切力，在每5秒一帧为5分钟（3-100达因/ 厘米 2），并记录图像中的应用。 </li><li>停止关闭注射泵的剪应力，获得2-3额外的图像，然后停止对软件的图像采集序列。 </li><li>接下来，删除第一个"退出"管，仔细，避免排空的通道，然后添加新鲜培养基预热，以填补之前删除"进入"管的井。 </li><li>为了定量确定的剪应力的作用，对细菌耐药性，可进行电镀实验如下:收集剩余的介质，并用120μL的PBS，删除剩余的介质通道（都洗两次的μ- SLIDE还收集了）。 </li><li>分离感染的细胞在37℃5分钟与50μL胰蛋白酶/ EDTA此外，在上一步所收集的暂停和混合分离样品。 </li><li>执行连续稀释在PBS和板10μL分数到GCB琼脂平板上，一式三份，以确定第二天的菌落形成单位（CFU）的板孵化后，在37 ° C的孵化器5％（V / V）的CO 2。 </li></ol> 影片3和4比较淠绿突变体与野生型菌株。 7。细菌支队从microcolonies <ol><li>感染细胞在流动腔重复步骤2.1至2.8，并允许进行30分钟的感染。 </li><li>一段2分钟的流量增加至10达因/ 厘米 2，删除未绑定的细菌，使感染继续为2h。 </li><li>减少流量0.15达因/厘米2。 </li><li>每隔一小时，收集流室，中型下降。 </li><li>执行系列稀释样品和GCB琼脂平板上板他们。 </li><li>确定第二天的菌落形成单位（CFU）的，板块的孵化后在37℃的孵化器C的5％（V / V）的CO 2 。 </li></ol> 8。代表性的成果<img alt="图1" src="/files/ftp_upload/3241/3241fig1.jpg" /> 图1:在流动过程中的存在，可以观察和测量的一个细胞单层的不同步骤。 <img alt="图2" src="/files/ftp_upload/3241/3241fig2.jpg" /> 图2:在实验（剪应力的情况下）开始在流动腔的内皮细胞单层。约50内皮细胞组织在子汇合单层。 <img alt="图3" src="/files/ftp_upload/3241/3241fig3.jpg" /> 图3:初步粘附细菌的细胞表面上的图形表示。三个字段的值是表示磁场对磁场的变化，预计（0044达因/ 厘米 2）的想法。 视频1。初始的细菌粘附细胞单层作为时间的函数（0044达因/厘米2）可视化。表达GFP的细菌以下的流动介质的方向。这部电影是加快了60倍，真正的视频持续时间为10分钟。 <a href="/files/ftp_upload/3241/3241video1.mov">点击这里观看视频。</a> 视频2，经过初步粘附的细菌。被允许细胞表面上的增殖期为7小时（加速1000倍）。 <a href="/files/ftp_upload/3241/3241video2.mov">点击这里观看视频。</a> 视频3。细胞表面上的机械师，后扩散剪应力水平增加5分钟，但野生型microcolonies耐加速60倍到10达因/厘米2人microcolonies阻力进行了测试。 <a href="/files/ftp_upload/3241/3241video3.mov">点击这里观看视频。</a> 由淠绿突变体形成的视频 4。Microcolonies被打乱流量的增加（10达因/厘米2 ）。这种突变失败诱导质膜下microcolonies重塑，从而更高度敏感，增加剪应力（视频加速60倍）5。 <a href="/files/ftp_upload/3241/3241video4.mov">点击这里观看视频。</a>

<ol>
	<li>Mairey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microcirculation+shear+stress+levels+determine+Neisseria+meningitidis+attachment+sites+along+the+blood-brain+barrier.">Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.</a> J Exp Med. 203, 1939-1950 (2006).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+Principles+of+Vascular+Beds.">Design Principles of Vascular Beds.</a> Circ Res. 77, 1017-1023 (1995).</li><li>Zarbock, A., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesion+and+activation+under+flow.">Neutrophil adhesion and activation under flow.</a> Microcirculation. 16, 31-42 (2009).</li><li>Tissot, O., Pierres, A., Foa, C., Delaage, M., Bongrand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+of+cells+sedimenting+on+a+solid+surface+in+a+laminar+shear+flow.">Motion of cells sedimenting on a solid surface in a laminar shear flow.</a> Biophys J. 61, 204-215 (1992).</li><li>Mikaty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+bacterial+pathogen+induces+host+cell+surface+reorganization+to+resist+shear+stress.">Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress.</a> PLoS Pathog. 5, e1000314-e1000314 (2009).</li><li>Chamot-Rooke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+modification+of+pili+upon+cell+contact+triggers+N.+meningitidis+dissemination.">Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.</a> Science. 331, 778-782 (2011).</li><li>Soyer, M., Dum&#237;nil, G., Christodoulides, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+under+shear+stress.">Bacterial adhesion under shear stress.</a> Neisseria meningitidis: Methods and Protocols (Methods in Molecular Biology). , (2011).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Thomas, W. E., Trintchina, E., Forero, M., Vogel, V., Sokurenko, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesion+to+target+cells+enhanced+by+shear+force.">Bacterial adhesion to target cells enhanced by shear force.</a> Cell. 109, 913-923 (2002).</li><li>Tchesnokova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-enhanced+binding+of+intestinal+colonization+factor+antigen+I+of+enterotoxigenic+Escherichia+coli.">Shear-enhanced binding of intestinal colonization factor antigen I of enterotoxigenic Escherichia coli.</a> Mol Microbiol. 76, 489-502 (2010).</li><li>Konto-Ghiorghi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+for+pilus+in+adherence+to+epithelial+cells+and+biofilm+formation+in+Streptococcus+agalactiae.">Dual role for pilus in adherence to epithelial cells and biofilm formation in Streptococcus agalactiae.</a> PLoS Pathog. 5, e1000422-e1000422 (2009).</li></ol>

没有利益冲突的声明。

剪应力，一般在生物力学方面的重要性正日益被人们所认识。例如高度适应胶粘剂的选择素家族的蛋白质在细胞粘附和血管壁的滚动过程中已确认的过程中通过引入剪应力。上文所述的程序，适用于革兰氏阴性菌的脑膜炎奈瑟菌，但应适用于广泛的病原体。也为其他​​病原体和其他感染部位的剪应力的重要性已被证明。细菌粘附剪应力的条件下已经FimH黏附研究发现肾盂肾炎大肠杆菌（UPEC） 9。类似选择素，被证明是由剪切引起的机械力和CfaE 肠毒素大肠杆菌黏附加强FimH和其宿主细胞受体之间的相互作用也有报道调解肠上皮细胞的粘附，通过剪切依赖性机制10。我们报道，使用的层流室，无乳链球菌菌毛为流动条件下的上皮细胞 11坚持这种病菌是至关重要的本章中描述的实验协议。这些研究证实，我们的流室法是一个有用的工具，调查宿主细胞病原体相互作用的剪应力条件下。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> μ- SLIDE VI 0.4流试剂盒</td><td> IBIDI </td><td> 80606 </td></tr><tr><td>注射器Plastipak 50毫升鲁尔锁</td><td>碧迪</td><td> 300865 </td></tr><tr><td>聚乙烯管材R3603 3.2 ×4.8毫米</td><td>费雪的科学</td><td> R3603 </td></tr><tr><td> 3路阀，2名女露儿男性露儿</td><td> Bio - Rad公司</td><td> 7328103 </td></tr><tr><td>注射泵</td><td>哈佛器械</td><td>博士2000 </td></tr><tr><td>倒置显微镜，尼康</td><td>尼康</td><td> Eclipse T I的</td></tr><tr><td> CCD相机</td><td>滨松</td><td> ORCA的285 CCD或ORCA的3 - CCD </td></tr><tr><td> ImageJ软件</td><td>国立卫生研究院</td><td>软件 （ <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> ） </td></tr></tbody></table>

introducing shear stress in the study of bacterial adhesion

在细菌感染的病原体及其宿主之间发生的相互作用序列。细菌粘附到宿主细胞表面往往是最初的发病机制和步骤。虽然大多在静态条件下研究实验附着力附着力实际上是在流动的液体的存在。细菌和他们的主人之间的第一次接触，经常会出现水平的粘膜，口腔，肺，肠，眼，等上皮细胞表面的粘液沿流动。后来，在感染，病原体偶尔访问的血液循环，导致危及生命的疾病，如败血症，败血症和脑膜炎。这些感染的一个定义特征是这些病原体与血管内皮细胞在血液循环的存在交互能力。流动的液体，粘液或血液，例如存在，决定粘连，因为它会产生对病原体的机械力。为了描述流动的液体之一的效果通常是指剪应力，这是一个固定的墙附近的一个移动流体，达因/厘米2表示每单位面积所施加的切向力的概念。剪应力强度有很大的不同根据不同船只的类型，大小，器官，位置等（0-100达因/厘米2）。毛细血管的循环，可以达到非常低的剪应力值，甚至暂时停止期，在几秒钟之间到几分钟1的。在频谱剪应力在动脉的另一端，可以达到100达因/厘米2。剪应力在不同的生物过程的影响已清楚地表明，例如在白细胞与内皮细胞的3的相互作用。要考虑到这种细菌粘附过程中的机械参数，我们利用了基于4室使用一次性流的实验过程。宿主细胞是生长在流室，荧光细菌注射泵控制流量介绍。我们最初集中在细菌病原体脑膜炎奈瑟菌 ，革兰氏阴性菌败血症和脑膜炎等负责调查。这里描述的步骤，使我们能够研究细菌的能力的剪应力的影响:坚持细胞1列，增殖和细胞表面上的5 分离殖民6（图1）新的站点。补充的技术资料中可以找到参考文献7。这里介绍的剪应力值分别为选择基于我们以往的经验，并表示在文献中找到的值。该协议应适用于广泛的病原体根据研究目标的具体调整。

Watch this Scientific Journal Video about Introducing Shear Stress in the Study of Bacterial Adhesion at JoVE.com

Introducing Shear Stress in the Study of Bacterial Adhesion

在感染过程中，一个关键的步骤是病原体与宿主细胞的粘附。在大多数情况下，粘附步骤发生在流动的液体所产生的机械应力的存在。我们描述了一种技术，细菌粘附的研究中的一个重要参数引入剪应力。

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 ...

introducing-shear-stress-in-the-study-of-bacterial-adhesion

Research

JoVE Journal

Immunology and Infection

15.6K 次观看.  INSERM U970. 在感染过程中，一个关键的步骤是病原体与宿主细胞的粘附。在大多数情况下，粘附步骤发生在流动的液体所产生的机械应力的存在。我们描述了一种技术，细菌粘附的研究中的一个重要参数引入剪应力。

视频: Introducing Shear Stress in the Study of Bacterial Adhesion

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 ...

科研

免疫与感染

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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 ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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 ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...