Name: application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells
Uploaded: 2011-12-17T14:00:00
Description: Watch this Scientific Journal Video about Application of a Mouse Ligated PeyerÃ¢â‚¬â„¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

作者想感谢所有欧洲投资银行的成员，为开发这一技术。这项研究得到了部分科研优先领域“交通膜”（议员）和感染现象“黑客帝国”（KH），青年科学家（SF和KH），科研（何的格兰特在援助）和“细胞内物流”（何）从教育部，文化，体育，科学和技术日本，创新领域的科学研究。

1。细菌细胞的制备<ol><li>摆放着数甘油荧光的细菌（如绿色荧光蛋白表达的鼠伤寒沙门氏菌）的LB琼脂平板，含100μg / ml的氨苄青霉素。 </li><li>文化从单菌落LB琼脂2毫升的新的LB培养基中过夜。 </li><li>添加0.5毫升细菌培养，直到在600 nm的光密度达到1.0至4.5毫升的新的LB培养基孵育。 </li><li>收获的细菌细胞，离心（3000 XG，5分钟，4℃）。 </li><li>弃上清，5.0毫升的无菌磷酸盐缓冲液（PBS）洗?...

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	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

<html>结扎Peyer氏菌悬液的补丁的孵化时间通常为1小时观察到M细胞的细菌掺入。在4小时孵化的情况下，细菌往往检测Peyer氏斑的T细胞区。由于蒸发异氟醚吸入麻醉小鼠保持稳定，结扎Peyer氏菌悬液的补丁的孵化时间可延长至观察T细胞Peyer氏补丁区移动的细菌。这个实验的重要的一点是要小心，不要划伤血管结扎肠道时。在不使用GFP表达的细菌的情况下，我们也可以选择使用商业荧光标记试剂荧光标...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> LB琼脂氨苄青霉素 100 </td><td>西格玛</td><td> L5667 </td><td></td></tr><tr><td> Cy3标记单反应 染料包</td><td> GE医疗集团</td><td> PA23001 </td><td></td></tr><tr><td>的Alexa Fluor 350 羧酸， 琥珀酰亚胺酯</td><td> Invitrogen公司</td><td> A10168 </td><td></td></tr><tr><td>吸入麻醉 仪器</td><td>信浓制作所</td><td> SN - 487 </td><td></td></tr><tr><td>固定和 通透 解决方案</td><td>屋宇署</td><td> 554722 </td><td></td></tr><tr><td>抗鼠 糖蛋白2 抗体</td><td> MBL </td><td>D278 - 3 </td><td> 200倍稀释液（5μg/ml） </td></tr><tr><td>羊抗鼠IgG， F（AB&#39;）2片段 具体</td><td>杰克逊 ImmunoResearch </td><td> 112-505-006 </td><td> 200倍稀释液（20μg/ml） </td></tr><tr><td>的Alexa Fluor 633 鬼笔环肽</td><td>分子探针</td><td> A22284 </td><td> 50倍稀释</td></tr><tr><td>激光共聚焦 扫描显微镜</td><td>徕卡显微系统</td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision 恢复 反卷积 显微镜</td><td>应用精密</td><td> DeltaVision核心</td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

我们的肠道内居住的共生细菌的数量巨大。对胃肠道的黏膜表面不断暴露给他们，并偶尔病原体。肠道相关淋巴组织（GALT）为诱导黏膜免疫反应，这些微生物1，2中发挥了关键作用。要启动黏膜免疫反应，粘膜抗原必须运从肠腔整个组织，如Peyer氏斑淋巴滤泡的上皮屏障。这介导的抗原transcytosis是由专门的上皮M细胞3， 4。 M细胞是非典型的上皮细胞，积极吞噬大分子物质和微生物。 M细胞与树突状细胞（DCs）和巨噬细胞，靶抗原降解的溶酶体，主要transcytose内在抗原。这种积极通过M细胞的大分子transcytosis提供抗原的根本组织淋巴滤泡D是认为启动抗原特异性黏膜免疫反应的关键。然而，促进M细胞抗原摄取的分子机制在很大程度上是未知的。我们曾报道，糖蛋白2（GP2），特别是肠之间的M细胞的根尖质膜上表示，作为一个transcytotic受体的共生和致病肠杆菌的一个子集，包括大肠杆菌和沙门氏菌血清型鼠伤寒沙门氏菌（鼠伤寒沙门氏菌），通过承认FimH，细菌外膜5菌毛类型的组成部分，我。在这里，我们提出了一个鼠标Peyer氏修补肠袢的检测，以评估M细胞的细菌摄取的应用方法。这个方法是鼠标肠袢先前所描述的6，7含量的改进版本。改进的要点如下：1。异氟醚被用作麻醉剂。 2。约1厘米结扎肠循环includING Peyer氏补丁成立。 3。 M细胞的细菌荧光标记的荧光标记试剂或过度表达的荧光蛋白，如绿色荧光蛋白（GFP）。 4。 M细胞相关的卵泡上皮覆盖Peyer氏补丁与反GP2抗体检测整个安装immunostainig。 5。荧光共聚焦显微镜分析观察M细胞的细菌transcytosis。小鼠Peyer氏补丁肠袢实验可以提供答案，什么样的共生或transcytosed M细胞的致病细菌，并可能导致我们了解如何通过M细胞刺激粘膜免疫系统的分子机制。

Watch this Scientific Journal Video about Application of a Mouse Ligated PeyerÃ¢â‚¬â„¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

在一个专门的卵泡相关Peyer氏斑上皮M细胞在肠道相关淋巴组织的粘膜免疫监视中发挥的重要作用。在这里，我们描述了细菌transcytosis M细胞的评价方法<em&gt;在体内</em&gt;。这种方法提供了一种方法，以了解在免疫系统中的M -细胞功能。

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

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JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Invasion of Human Cells by a Bacterial Pathogen

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

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 ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

的视觉检测，以监测T6SS介导的细菌竞争

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长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

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检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

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单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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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 ...

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一种新的体外创面愈合试验评价细胞迁移

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