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，根据机构动物护理和使用委员会（IACUC）的指导方针。 </li><li>用70％乙醇或优碘，手术前清洗腹部。执行标准剖腹手术，涉及到一个单一的（1厘米）切口手术级的剪刀沿中线开始从基地约1.5厘米的肋骨。公开每itoneal腔，并确定盲肠。剪断终端小肠回肠盲肠交界处，轻轻取出肠道内的全部。 ，请注意不要超伸被删除，因为它是从腹膜腔肠组织。 </li><li>在床上的湿纸巾或Kimwipes奠定小肠。湿用生理盐水轻轻的小肠，以防止组织脱水。可视地识别个别位于肠道反肠系膜侧（ 图1）的PP。通常情况下，一个单一的鼠标具有均匀分布从十二指肠（近端）回肠（远端）的5至10可见的PP。平均而言，四只老鼠将产生23的PP。 </li><li>使用弯曲的手术剪刀，轻轻消费个别PP和将它们放置在冷的Hank氏平衡盐溶液（HBSS）。 笔记 ，剪刀应该被放置曲线侧的正上方的PP，然后轻轻地施加到组织。海关关长只的PP（surroundi的异常组织），转移到冷的HBSS。 注:所有的孵化和离心步骤，这一点在第2节，应在4°C保存细胞活力。 </li><li>转移的PP5毫升健脾解离中孵育15-20分钟，在37°C，同时大力摇晃在250转。培养时间的延长细胞活力将产生负面影响。 </li><li>要产生单细胞悬浮液，无菌（蒸压）为70μm的尼龙网细胞过滤网上的地方的PP和强行研磨组织成网状，使用从1毫升注射器的柱塞的基。另外，PPS磨砂无菌玻璃显微镜幻灯片（高压灭菌信封），轻轻地磨一个来回运动组织之间的夹层。使用无菌移液管转移到一个5毫升的Falcon管中的细胞悬浮液。 </li><li>加入EDTA至终浓度为1mM的细胞悬浮液。孵育摇杆在室温下5分钟。 </li><li>倒出细胞悬液通过第二个70微米细胞过滤器，以消除任何剩余的细胞聚集或组织碎片。收集细胞中有15或50毫升锥形管。 </li><li>主题细胞温和离心（在500×g离心5分钟）。在流的缓冲液，磷酸盐缓冲盐水（PBS）含有1％胎牛血清（FCS）的滗析上清液和重悬细胞。 </li><li>为了确定细胞数和存活率，稀释1:10到台盼蓝染色的细胞，并目视检查用光学显微镜和血细胞计数仪的细胞。可替换地，一个的伯爵夫人细胞计数器（Invitrogen公司），或类似的仪器，可以使用自动枚举细胞数和生存能力。 20-25 PPS开始，这个协议应该产生2.5×10 6个细胞总数。这相当于〜0.8-1.2×10 6细胞每只小鼠。 </li></ol>抗体标记的细胞用于流式细胞仪 &lt;OL开始="11"&gt; <li>分送成的96孔圆底板的孔中的细胞（每孔10 5）。 </li><li>主题板温和离心（在1,000 xg离心5分钟），并倾析上清液轻轻板反转。 </li><li> FC块缓冲液重悬细胞，并在冰上孵育15分钟。虽然有许多市售的FC块缓冲区（ 如:大鼠抗小鼠CD16/CD32，BD Biosciences公司），我们简单地使用花介质从老鼠的B细胞杂交瘤（ATCC 2.4.G2）分泌的单克隆抗体对小鼠Fcγ受体，此步骤。 </li><li>主题板温和的离心分离（在1000×g离心5分钟）以上，并倾析上清液轻轻板反转。 </li><li>直接加入荧光标记的抗体所需稀释的细胞悬浮液，冰上孵育30分钟，连续摇摆。 </li><li>主题板温和离心（在1,000 xg离心5分钟），并倾析上清液轻轻翻转板。 </li><li>用100微升的流动缓冲液洗涤细胞。 </li><li>离心板在1,000 xg离心5分钟，并倾析上清液轻轻板反转。 </li><li>重悬细胞加入400μl固定缓冲液，其中包括流缓冲液以300μl和100μl1％多聚甲醛在PHEM缓冲液（60 mM的筒，25mM的HEPES，10mM的EGTA，在pH = 6的MgCl 2 4mM的）。 </li><li>主题细胞，流式细胞仪使用FACSCalibur或同等学历。分析结果使用Cell任务Pro软件5.2版本。 </li></ol> 3。 PP冰冻切片的制备<ol><li>在推进组织收集，最佳切削温度（OCT）化合物，7x7x5毫米的塑料底座模具。还要准备一个游泳池（750毫升）的液态氮杜瓦瓶或冰桶中。 </li><li>安乐死鼠标和执行laparotamy，如上所述。删除的小肠和地方上湿纸巾。 </li><li>对隔离为cyrosectioning的PP，横向切0.5厘米段的小肠内含有一个单一的PP和转移组织培养皿中包含PBS。这些段与PBS轻轻冲洗管腔，以消除不必要的粪便杂物。 </li><li>将肠组织分部内垂直基地模具OCT。浸没在液氮中的模具，并允许组织完全冻结（1-2分钟）。在OCT会改变颜色，从透明至白色，当组织被完全冻结。对于一个逐渐冷却（ 例如 ，减少开裂的OCT），浸泡在用液氮蒸气浴冷却的异戊烷的模具。 注意:穿戴合适的工作时，用液态氮的安全护目镜。 </li><li>从液氮使用钳子删除已冻结的基模具，并允许模具预热只是足够长，在室温下（约10-15秒），以允许在OCT块的边缘软化。然后删除冻结块OCT倒置的模具的模具，并在后面轻轻按下，退出块到一个干净的4×4 cm长的铝箔。立即在箔片和地点在液氮或干冰上存储的标记的试样袋包裹组织。或者，转移组织中的冷冻器（&lt;-20℃）。使用组织在一个星期内，否则，块随着年龄的增长会变脆。 </li></ol> Cryosectioning <ol start="6"><li>冰冻切片的组织使用一个徕卡CM3050S的冷冻切片机或同等学历。应设置在低温恒温器的腔室的温度为-21℃。 </li><li>争取是12-14微米厚的部分。 </li><li>费舍尔的Superfrost Plus（或同等学历）显微镜玻片上收集部分和视觉使用的是标准的低功耗光学显微镜检查。如果有多个部分正在收集有关的幻灯片，确保他们聚集在一起下游染色应用。 </li><li>商店吨他滑动的滑动框，并在室温下在理想的情况下，使用它们在几天之内。 </li></ol>冰冻切片抗体标记和共聚焦显微镜分析<ol start="10"><li>浸入丙酮（或甲醇）的组织染色缸或机架2分钟的幻灯片。长时间的潜伏期，可能会导致组织中分离的幻灯片。 </li><li>转移到新的染色缸的幻灯片，并用PBS-T（1X用0.05％Tween-20的PBS）洗3次，每次3分钟。 </li><li>与的ImmEdge疏水笔包围的部分。这将确保试剂和抗体将保持期间只限于该区域孵育。 </li><li>在一个加湿室中在37℃下30分钟，避光孵育在块缓冲液（2％山羊血清的PBS）的幻灯片。 </li><li>孵育10分钟的FC块缓冲区（见上文）的幻灯片，在37°C。 </li><li>浸在PBS-T和干燥的部分使用Kimwipes或其他吸水组织周围地区的幻灯片。小心不要接触到实际的组织切片。 </li><li>叠加组织切片与初级抗体溶液并孵育30分钟，在37℃下的潮湿室中。初级抗体通常是在块缓冲液稀释至终浓度为20微克/毫升。直接标记的第一抗体的使用是可取的，直接在此步骤中，因为多达三个抗体可以结合。直接标记的抗体的同时也消除了需要额外的抗体孵育步骤。 </li><li>浸泡在PBS中洗涤3次（每次5分钟）的滑动。 </li><li>如果必要，孵育与有关的二次抗体，在37℃下30分钟，在湿气室内滑动。 </li><li>浸泡在PBS中洗涤3次（每次5分钟）的滑动。 </li><li>孵育4分钟，在1％多聚甲醛在PHEM缓冲液中，如上文所述的幻灯片。 </li><li>冲洗1分钟，用PBS滑动。 </li><li>干周围地区的部分使用Kimwipes或其他吸收耳鼻喉科组织。小心不要接触到实际的组织切片。使用移液管或滴管轻轻将延长黄金直接安装介质上的部分下降。应用盖玻片上的安装介质照顾，以避免气泡。用吸水纸，以吸收任何多余的安装介质。 </li><li>密封盖玻片显微镜载玻片与商业的指甲油，并在空气中晾干。 </li><li>查看幻灯片用Leica TCS SP5或有同等作用的共聚焦显微镜。 </li></ol>

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没有利益冲突的声明。

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％FCS?...

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 ...

isolating-immunostaining-lymphocytes-dendritic-cells-from-murine

Research

JoVE Journal

Immunology and Infection

32.5K 次观看.  New York State Department of Health. 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。

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

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

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

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 ...

科研

免疫与感染

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

从小鼠的补丁中提取体液免疫的关键参与者: 先进和优化的淋巴细胞分离协议

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

癌症研究

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

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 presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

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 ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

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

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified 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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the 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 ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

从小鼠佩耶的补丁中对Germinal中心B细胞JH4因特龙的体细胞突变分析

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...