作者要感谢阿兰博士Trautmann鼓励我们进行淋巴结切片。这项工作是支持部分由法甲国立CONTRE LE癌症基金会POUR LA RECHERCHE Médicale EN法国和协会LA RECHERCHE河畔乐癌症的赠款。

1。从淋巴结准备片<ol><li>准备4％低熔点琼脂糖溶液嵌入。琼脂糖稀释在PBS和微波这个解决方案。当琼脂糖完全溶解，保持在37 ° C，直到准备使用的解决方案。 </li><li>准备一个6孔组织培养板。加入1.1毫升每口井的RPMI完全培养液，并插入在每口井器官的过滤器。 6孔板放置在4 ° C，直到准备转让片。 </li><li>广场不锈钢垫圈，内径为4毫米，为填充用RPMI完全培养基一个塑料盘。垫圈将进一步集中vibratome切割片上的细胞。 </li><li>鼠标在根据当地的动物福利法规牺牲。小心地从周围组织的外周淋巴结和他们在一个塑料盘含冰冷PBS的地方。必须小心不损坏淋巴结结构，因为这些器官都非常柔软。 </li><li>倒入一个35毫米的塑料盘的4％琼脂糖凝胶电泳和微妙的凝胶转移到节点。留在冰上5分钟硬化的琼脂糖凝胶。 </li><li>从盘中取出块，并以凝胶3-5毫米左右离开每个淋巴结修剪琼脂。 </li><li>附加标本无毒组织胶vibratome磁盘上的嵌入式节点。安装在弥漫着冰冷的PBS托盘的标本磁盘。 </li><li>科琼脂嵌入式vibratome一个缓慢的范围上设置的速度（0.3毫米/秒）和一个中程（1.5毫米）上设置的振动频率在320μm厚的组织。 </li><li>小心转移淋巴结切片，因为他们正在削减到的器官文化，用细镊子插入。放置在每个插入3片。使用非常谨慎，作为片可以很容易损坏。一个典型的周围淋巴结将削减产量5节时，在320微米。放弃第一个和最后一个切片，因为它们只包含浅表组织。 </li><li>每一个人的片上放置不锈钢垫圈。确保垫圈定位以及对周围组织琼脂糖。 </li><li>培养板在37 ° C直到准备板T细胞在5％ 二氧化碳培养箱。 </li></ol> 2。隔离从小鼠淋巴结T细胞<ol><li>根据先前公布的朱庇特的第3条隔离T细胞。 </li><li>立即使用的T细胞，并继续下一节。否则，可能是淋巴细胞培养过夜在10 ng / ml的IL - 7为辅的完整的RPMI介质。 </li></ol> 3。孤立的T细胞的标签<ol><li>在HBSS中清洗细胞。重悬在每毫升10 × 10 6相同的缓冲区。 </li><li>加入相同体积的含有1微米的细胞跟踪绿色CMFDA给予终浓度为0.5微米的HBSS。 </li><li>在37 ° C在5分钟的细胞悬液。完全RPMI培养基洗涤细胞两次。 </li><li>在终浓度为每毫升10 × 10 6完全RPMI培养基悬浮细胞。这些标记的细胞，将被镀上的切片。 </li></ol>比CMFDA其他荧光染料可能被使用过，但要记住，这些分子有可能超过一定浓度的有毒。 4。电镀标记的T细胞到淋巴结切片<ol><li>用移液器洗衣机的内部部分中除去多余的介质。不要让组织变得干燥;工作迅速在这一步。 </li><li>轻轻免除10％至20μL标记的T细胞，对应于1至2 × 10 5细胞在洗衣机内的一部分。必须小心，不要触摸吸管尖的组织。确保停留在细胞悬液下降。 </li><li>放置培养箱中培养细胞切片在37 ° C和5％的CO 2至少30分钟，让淋巴细胞迁移到组织。 </li></ol> 5。成像T细胞在淋巴结切片在这个视频文章中，我们用广角倒置显微镜和共聚焦直立显微镜的淋巴结切片图像荧光T细胞。 <ol><li>温度设定在37 ° C。我们的显微镜都配有温度控制箱。 </li><li>安装灌注系统，能够连续灌注含氧（5％CO 2，O 2的95％）无酚红的RPMI培养基的淋巴结切片。在我们的系统，灌流媒体从一个侧面受重力进入到成像室。介质与泵的连接到一个废物收集瓶中吸气。设置媒体流速为1毫升/分钟。 </li><li>用细镊子，微妙拿出从6孔板切片，浸在回暖含氧的RPMI中等几秒钟，以获得摆脱不渗透进入组织的荧光细胞的准备工作。 </li></ol> IMA蔓茎与T细胞的一个宽领域的倒置显微镜<ol start="4"><li>将片上粘到玻璃底部盘内的尼龙线组成的一个特制的成像室倒挂。在这个会议厅内，尼龙线提升玻璃切片，使氧扩散到组织的解决方案。淋巴结的T细胞迁移是强烈地依赖氧气，这是必需的。安全切片，加入到它的不锈钢垫圈。在这种情况下，切片在于以上的菜，这有利于延续灌注液底部几微米。 </li><li>要捕获的大视场（800 × 800微米，一个节点的片表面的约三分之一），采集图像，使用10倍的干物镜。我们发现，尼康10X小号福陆公司0.5 NA是最适合我们的分析。 </li></ol>一个典型的时间推移成像实验捕获5，涵盖了总深度在轴向（Z）尺寸50微米的光飞机。荧光T细胞通常成像范围在10至20分钟10到30秒的时间间隔。 直立共聚焦显微镜成像T细胞在本议定书中所使用的激光共聚焦显微镜是徕卡配备20倍水浸泡的目的（奥林巴斯，20x/0.95 NA）的SP5的。 <ol start="6"><li>片固定在显微镜的成像阶段。注：我们通常放在编制固定垫圈。 </li><li>设置收集了一系列的图像沿Z轴的成像会议。在一个完整的组织结构图像的T细胞，起始位置通常是在下面的第一个标记的T细胞的5至10微米。 </li></ol> 6。代表性的成果通过下面这个视频协议，你应该预料到一个节点的T区，这种现象通常发生在这特定的环境中体内（图1）中积累的荧光性T细胞的大量可视化。尤其是T细胞，B细胞区，下方胶囊通常应排除。一个成功的实验，也将导致进入组织招募的T细胞（图2），显示高度能动的行为（图3）与公布的结果获得完整的淋巴结肿大4一致。平均而言，片内的个人T细胞的平均流速应接近10微米/分钟（图4）。 <img src="/files/ftp_upload/3054/3054fig1.jpg" alt="图1"/> 图1。 T细胞聚集在淋巴结切片。淋巴结切片图像采集前30分钟（上图片），分别加入荧光标记的T细胞（CMFDA绿色） 。形象是最大的5跨越50微米，在z方向的图像投影。明场图像显示在底部。与宽视场显微镜图像捕获。 <img src="/files/ftp_upload/3054/3054fig2.jpg" alt="图2"/> 图2。 T细胞被招入一个淋巴结切片 ，淋巴结切片图像采集前30分钟，使用共聚焦显微镜，分别加入荧光标记的T细胞（CMFDA绿色）。在切割面（上方照片）和40微米以下（下图）的图像被抓获。 <img src="/files/ftp_upload/3054/3054fig3.jpg" alt="图3"/> 图3。 T细胞是高度能动内淋巴结切片 ，荧光标记的T细胞在12分钟用一个宽视场显微镜成像。个别T细胞的轨迹显示颜色编码的轨道，代表从蓝色（低游动细胞），红色（高游动细胞）增加位移。轨道计算Imaris软件。白线以下的边缘节点，而虚线椭圆划定的公认的B细胞区。 <img src="/files/ftp_upload/3054/3054fig4.jpg" alt="图4"/> 图4：在一个淋巴结切片，T细胞的速度接近10微米/分钟。从图3中的代表曲目Imaris软件计算的速度。

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

我们已经描述了一个简单，快速和强大的技术产生淋巴结切片，这是用来调查推出的T细胞的行为。近年来，这种方法已经被成功应用胸腺和淋巴结切片识别控制T细胞的定位和运动1,5的细胞外因素。它也被用来在积极选择和T细胞抗原识别2,6时的胸腺细胞的Ca 2 +反应来衡量。覆盖切片检测的优势和局限，值得讨论。值得注意的是，这个系统允许访问的组织，有用的，如果需要操纵片药理学，以干扰与细胞迁移的分子控制。成像后，可切片进行免疫组化处理，以收集对已成像结构的进一步信息。此外，可与传统的宽视场荧光显微镜观察。虽然分辨率不如共聚焦或双光子显微镜，光毒性，原则上是更严重的单光子双光子显微镜，它具有简单的优势，以较低的成本和更大的选择，激发波长。 一个完整的淋巴结中的T细胞成像需要静脉注射标记的淋巴细胞，淋巴器官，随后回家。正如我们以前1所示，切片化验继转移实验是完全兼容的。然而，T细胞进入淋巴结家庭所需的时间长度，可以使用荧光染料，有一种倾向，泄漏的细胞，随着时间的推移变得复杂。这是的Ca 2 +染料FURA - 2的情况下。快速招聘到组织的T细胞（&lt;30分钟），切片检测提供了一个机会使用这些染料。最后，这种方法具有很大的优势分析在几个人体组织中的T细胞功能，更让。 这个实验系统介绍也需要牢记的限制。与切片相关的损害可能影响T细胞的功能，尤其是在表面的切割面附近的组织区域。为了评估在切片的细胞​​死亡，我们用荧光染料SYTOX绿色，穿透细胞受损的细胞膜。我们的实验表明，约20％，总的淋巴结细胞，大多定位在肤浅的地区组织，这个核染料荧光标记。检测的另一个潜在的问题是能否留住重要的可溶性因子包括趋化因子的切片。虽然，我们没有任何迹象显示，我们的数据等问题​​的影响，因为T细胞内片显示良好的蠕动，我们想强调的成像T细胞在位于从切面的几十微米的健康地区的重要性。然而，这可能是与传统的显微镜，如在本议定书（广角或焦），它是可能的淋巴结切片准备，结合双光子成像深度将增加空间分辨率和降低光毒性。

<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>共聚焦显微镜</td><td>徕卡</td><td> SP5 </td><td></td></tr><tr><td> Vibratome </td><td>徕卡</td><td> VT1200S </td><td></td></tr><tr><td>精细镊子</td><td>世界精密仪器</td><td> 14142 </td><td></td></tr><tr><td> 30毫米的文化插入</td><td> Millipore公司</td><td> PICM0RG50 </td><td></td></tr><tr><td>的RPMI </td><td> Invitrogen公司</td><td> 61870010 </td><td>完全RPMI培养基加入10％热灭活小牛血清和青霉素/链霉素</td></tr><tr><td>汉克斯“平衡盐溶液（HBSS） </td><td> Invitrogen公司</td><td> 14170088 </td><td></td></tr><tr><td>低胶凝温度琼脂糖，类型七- A </td><td> Sigma - Aldrich公司</td><td> A0701 </td><td></td></tr><tr><td>氰基丙烯酸正丁酯胶，Vetbond </td><td> 3M </td><td> 1469 </td><td></td></tr><tr><td>不锈钢垫圈，内径4毫米</td><td> DIY店</td><td></td><td></td></tr><tr><td>细胞跟踪绿色CMFDA </td><td> Invitrogen公司</td><td> C7025 </td><td></td></tr></tbody></table>

ex vivo imaging of t cells in murine lymph node slices with widefield and confocal microscopes

幼稚T细胞不断交通次级淋巴器官，包括周边的淋巴结，发现罕见表达的抗原。 T细胞迁移到淋巴结是一个复杂的过程，它涉及包括趋化因子的细胞和化学因素。近日，双光子显微镜的使用已获准跟踪完整的淋巴结中的T细胞，并从中对他们的行为和它们之间的相互作用的一些定量信息与其他细胞。虽然有明显的优势，在体内系统，这种方法需要一个复杂的和昂贵的仪器和组织提供有限的访问。要分析小鼠淋巴结中的T细胞的行为，我们已经开发出一个切片检测1，最初成立由神经生物学家最近调换到小鼠胸腺2。在这种技术中，荧光标记的T细胞被镀上敏锐地准备的淋巴结切片的顶部。在这个视频，文章，本地化和T细胞迁移到组织中的实时分析与广角和共聚焦显微镜。技术，补充体内的双光子显微镜图像的T细胞提供了一个有效的方法在自然环境中，并阐明T细胞迁移的内在机制。

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

这个协议描述了一种方法引入到淋巴结切片的荧光图像的T细胞。该技术允许传统的宽视场荧光或共聚焦显微镜的实时分析，T细胞迁移。

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

ex-vivo-imaging-t-cells-murine-lymph-node-slices-with-widefield

Research

JoVE Journal

Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

16.7K 次观看.  Université Paris Descartes, CNRS (UMR 8104). 这个协议描述了一种方法引入到淋巴结切片的荧光图像的T细胞。该技术允许传统的宽视场荧光或共聚焦显微镜的实时分析，T细胞迁移。

视频: Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

小鼠浅表淋巴结手术

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

淋巴结脂肪垫对嵌合体的淋巴结基质细胞起源研究的一代

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...