Name: isolating and immunostaining lymphocytes and dendritic cells from murine peyer's patches
Uploaded: 2013-03-17T14:00:00
Description: Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

我们感谢狄仁杰歌（Wadsworth中心流式细胞仪核心）协助细胞分析和海伦·约翰逊（Wadsworth中心动物病理组织核心）的石蜡切片的制备。我们感谢理查德·A·科尔博士（Wadsworth中心光学显微镜核心），激光共聚焦显微镜和图像采集的援助。我们要感谢安迪·本特利（沃兹沃斯中心的照片和插图）与动画的援助。 MDJ支持生命科学研究基金会，霍华德·休斯医学研究所（HHMI）的奖学金。 SA的支持，一个沃兹沃斯中心健康研究公司壁间的博士后研究。这项工作是支持的，部分由美国国立卫生研究院资助HD061916和GM082978。

根据传统，无特定病原体动物被安置和处理沃兹沃斯中心的机构动物管理和使用委员会（IACUC）的指导方针完全符合。 1。口灌胃<ol><li> （可选）灌胃小鼠品系的选择与抗原或微生物使用X1.5-22 G的兴趣。钝端喂食针（，波普尔科学，海德公园，NY）。交货量不应超过400微升每鼠标。 </li></ol> 2。 PP细胞用于流式细胞仪分离<ol><li>安乐死小鼠CO 2窒息，?...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peyer's+Patch+Dendritic+Cells+Sample+Antigens+by+Extending+Dendrites+Through+M+Cell-Specific+Transcellular+Pores.">Peyer's Patch Dendritic Cells Sample Antigens by Extending Dendrites Through M Cell-Specific Transcellular Pores.</a> Gastroenterology. 42, 592-601 (2011).</li><li>Lelouard, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+bacteria+and+dead+cells+are+internalized+by+a+unique+subset+of+Peyer's+patch+dendritic+cells+that+express+lysozyme.">Pathogenic bacteria and dead cells are internalized by a unique subset of Peyer's patch dendritic cells that express lysozyme.</a> Gastroenterology. 138, 173-184 (2010).</li><li>Shreedhar, V. K., Kelsall, B. L., Neutra, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+induces+migration+of+dendritic+cells+from+the+subepithelial+dome+region+to+T-+and+B-cell+areas+of+Peyer's+patches.">Cholera toxin induces migration of dendritic cells from the subepithelial dome region to T- and B-cell areas of Peyer's patches.</a> Infect. Immun. 71, 504-509 (2003).</li><li>Favre, L., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+IgA+possesses+intrinsic+modulatory+properties+stimulating+mucosal+and+systemic+immune+responses.">Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses.</a> J. Immunol. 175, 2793-2800 (2005).</li><li>Kelsall, B. L., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+dendritic+cells+are+present+in+the+subepithelial+dome+and+T+cell+regions+of+the+murine+Peyer's+patch.">Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch.</a> J. Exp. Med. 183, 237-247 (1996).</li><li>Fukuda, S., Hase, K., Ohno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+Mouse+Ligated+Peyer's+Patch+Intestinal+Loop+Assay+to+Evaluate+Bacterial+Uptake+by+M+cells.">Application of a Mouse Ligated Peyer's Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells.</a> J. Vis. Exp. (58), e3225 (2011).</li><li>Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. 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在这篇文章中，我们提供了并行协议的准备PP准备流式细胞仪和功能分析和冰冻切片免疫组化染色的单细胞。这两种方法都高度重复性和容易获得，流式细胞仪和低温恒温器。对于第一次调查应该指出，当脾，总的电池产量PPS是相对贫乏。然而，该协议，概述一般为0.8〜1.2×10共6个 PP细胞从一个单一的鼠标之间的收益率。规模化是可能的，但我们通常不超过20-25 PPS池，因为，在我们的经验?...

Peyer氏修补程序（PPS）是宏观集合体有组织淋巴滤泡整个的小小肠人类和小鼠（ 图1）中存在的和构成的主站点在粘膜免疫应答被发起针对膳食抗原共生细菌，微生物病原体，口服疫苗1-4。与其他外周淋巴组织，如肠系膜淋巴结，PPS缺乏传入的淋巴管。因此，适应性免疫反应的PP中的驱动响应于来自肠腔抗原。管腔抗原的采样来完成的由卵泡关联的上皮（FAE），其中包括被称为M细胞肠细胞和抗原采样单元。上皮子圆顶（SED）的区域中，位于下方的FAE，间杂与巨噬细胞，B细胞和CD4 + T细胞5-9的树突状细胞（DC）的网络。在每个PP淋巴follicl的核心e是滤泡树突状细胞（FDCS）和B细胞丰富的中央生发中心，两侧的T细胞丰富的滤泡区。的PP的查询结果中的发展型IgA + B细胞浆母和CD4 +效应和记忆细胞抗原采样种子周围的固有层，并提供很宽的范围内的免疫力粘膜入侵者。 解剖复杂的免疫抗原采样，处理和呈现在PPS相关的事件，是一项艰巨的任务，考虑到PP细胞只占一小部分的总淋巴样细胞在肠黏膜。为了有助于在体外表征细胞在这样的环境中，我们提供了一个协议，用于制备总鼠标PP细胞流式细胞仪和功能分析，以及用于制备聚丙烯的冷冻切片的免疫荧光显微镜和免疫组化检测的协议。我们的分离，鉴定和免疫组织化学的谅解备忘录的协议ËPP细胞本身并不新颖，所证明的事实，即有超过25年的历史，多次提到，引用这些技术5,6,9-11。相反，我们的协议提供了一个简化的（视觉）的方法收集PPS第一次调查。我们所描述的技术很容易掌握，容易产生大量的细胞与细胞活力&gt; 90％。 cryosectioning协议产生高度重复的连续切片进行免疫荧光染色和激光共聚焦成像非常适合。此外，我们的协议补充其他两个最近的朱庇特的文章。第一，由福田和他的同事，描述了使用结扎回肠环路检测，以评估的致病细菌的吸收，由PP M细胞12。其他的，的GEEM和他的同事，介绍了小鼠肠黏膜的区议会和巨噬细胞的分离和鉴定，但明确排除了PPS从他们的分析13。

<table border="1"><tbody><tr><td> 项目 </td><td> 公司 </td><td> 猫。 ＃ </td><td> 评论（可选） </td></tr><tr><td> OCT复合</td><td>组织康</td><td> 4583 </td><td></td></tr><tr><td> 7x7x5 mm底座模具</td><td> Fisherbrand </td><td> 22-363-552 </td><td></td></tr><tr><td> ImmEdge笔</td><td>矢量实验室</td><td> H-4000 </td><td></td></tr><tr><td>的Superfrost Plus幻灯片</td><td> Thermo Scientific的</td><td> 4951 </td><td></td></tr><tr><td>边爱色丽刀片</td><td> Thermo Scientific的</td><td> 4280L </td><td></td></tr><tr><td> ANTI-MOUSE的CD11c-PE </td><td> eBioscience公司</td><td> 17-0114-82</td><td></td></tr><tr><td>反鼠标CD45R/B220-APC的</td><td> BD Pharmigen </td><td> 553092 </td><td></td></tr><tr><td> ANTI-MOUSE CD3-FITC </td><td> BD Pharmigen </td><td> 561798 </td><td></td></tr><tr><td>抗小鼠CD4-PE </td><td> BD Pharmigen </td><td> 553652 </td><td></td></tr><tr><td>抗小鼠CD8-PE </td><td> BD Pharmigen </td><td> 553032 </td><td></td></tr><tr><td>抗鼠CD19-PercP </td><td> BioLegend </td><td> 115531 </td><td></td></tr><tr><td> Hank氏平衡盐溶液（HBSS）不含有酚红</td><td> Fisher Scientific则</td><td> 14175-079 </td><td></td></tr><tr><td> 70微米的细胞过滤器</td><td> BD猎鹰</td><tD&gt; 352350 </td><td></td></tr><tr><td>健脾解离中</td><td>干细胞技术</td><td> 7915 </td><td></td></tr><tr><td>山羊血清</td><td> Invitrogen公司</td><td> 16210-072 </td><td></td></tr><tr><td> FC块</td><td> ATCC 2.4.G2 </td><td> HB-197 </td><td>从细胞线2.4.G2的Supes获得</td></tr><tr><td>弯剪式</td><td> FST </td><td> 14061-09 </td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> 3050S </td><td></td></tr><tr><td> FACS刀魂</td><td> BD </td><td></td><td></td></tr><tr><td>伯爵夫人细胞计数</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td>苏木</td&gt; <td>理查德·艾伦</td><td> 7211 </td><td></td></tr><tr><td>曙红</td><td>理查德·艾伦</td><td> 71304 </td><td></td></tr><tr><td>福尔马林</td><td> Starplex科学</td><td> 3661 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。本研究中所用的试剂和设备。 </td></tr></tbody></table>

isolating and immunostaining lymphocytes and dendritic cells from murine peyer's patches

Peyer氏斑（PPS）的肠道相关淋巴组织（GALT）是不可或缺的组成部分，并在肠道的免疫监视和平衡中发挥核心作用。连续采样在肠腔内的颗粒性抗原和微生物PP M细胞在卵泡相关上皮（FAE），运到底层网络的树突状细胞（DC）的，巨噬细胞，和淋巴细胞。在本文中，我们描述了小鼠的PP是（i）解离成单细胞悬浮液，并进行流式细胞仪，及（ii）用于cryosectioning和免疫制备的协议。流式细胞仪，PPS的机械分离，然后通过70微米膜过滤产生单细胞悬液的上皮细胞和大的杂物。用20-25的PP（从四只小鼠）开始，此快速和可重复性的方法产生一个人口&gt; 2.5×10 6细胞&gt; 90％的细胞存活率。对于cryosectioning，新鲜LY孤立的PP都沉浸在最佳切削温度（OCT）中，在液氮快速冷冻，然后切片用冷冻切片机。组织切片（5-12微米），空气干燥，用丙酮或甲醇固定，然后进行免疫标记。

单分散悬浮液的总PP细胞的流式细胞仪分析揭示了一个明确的区分好的和坏的细胞制剂。在良好的细胞制剂中，有超过80％的可行性，绝大多数的细胞表现出高的前向散射（FSC），细胞体积高的指标，以及低侧散射（SSC），低的细胞粒度（ 图2A）的指标。在这个实验中，我们还特意准备了一个“细胞制剂差”孵育PP细胞在室温下（而不是在冰上）在分离步骤，并在最后一步，用PBS加1％F...

Watch this Scientific Journal Video about Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches at JoVE.com

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

理解在派伊尔氏淋巴集结的特定的细胞亚群（PPS），初级感应网站肠相关的淋巴组织的免疫功能有越来越大的兴趣。在这里，我们列出了流式细胞仪分析和PP冰冻切片免疫准备的PP单细胞准备的并行协议。

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Research

JoVE Journal

Immunology and Infection

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

科研

免疫与感染

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

在体外产生的常见淋巴祖使用AC-6馈线系统的小鼠浆细胞样树突细胞

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...