我们感谢西尔维·为该协议的的关键的读数的勒萨热和的所有劳顾会委员。这项工作被受支持的由一个从的作者：卫生研究所（CIHR）的的加拿大研究院的的授出日期（澳门币-77545）。纳萨莉拉布雷克被由一个FRSQ的高级奖学金的的支持，，和MELISSA的Mathieu收到1 NSERC亚历山大·格雷厄姆·加拿大贝尔公司研究生奖学金。

1。手术前的准备工作根据加拿大动物护理准则理事会处理小鼠。 <ol><li> anesthetise小鼠注射氯胺酮/甲苯​​噻嗪（150/300毫克/千克，IP）或异​​氟醚吸入（2％，1L氧气）。如果可以，异氟醚应该使用，因为它允许为更好地控制麻醉时间和深度。此外，氧管理的同时，支持动物的生理功能。此外，异氟醚直接消除肺部，因此并不需要肝，肾代谢。 </li><li>注入丁丙诺啡（0.05 - 0.1毫克/千克，SC）或另一种止​​痛药。 </li><li>适用于鼠标的眼睛上的药膏，手术前，以避免眼睛干涩（如果使用异氟醚）。 </li><li>确认鼠标是睡着了捏它的爪子，滚它的尾巴。如果鼠标没有反应，进行手术。 </li></ol> 2。手术<p类=“jove_content”&gt;很可能浅表淋巴结（LN），通过手术移除4：2腹股沟和2臂丛。 LN的隔离更容易在年轻瘦老鼠，因为在老年小鼠的脂肪组织的存在（6-8周）可与LN隔离干扰。然而，这一限制被克服，反复练习。为的小鼠舒适，我们收获一次最多2淋巴结，当我们执行时间课程实验，我们收获LNS每隔一天。此外，为防止感染，抗生素可以给鼠标，但我们不这样做，伤口愈合得很好。一个需要注意，根据实验设计，使用抗生素，可能会影响观察的结果（例如，如果细菌的反应测量）。请注意，所有仪器每次使用后消毒和无菌条件下保持整个程序。 <ol><li>鼠标放在其副作用。 </li><li>本地化的地区，在那里你会执行incisioN，这取决于你想收获的法律公告。 （ 图1）。 </li><li>洗必泰，或另一种消毒剂，适用于该地区。 </li><li>请用锋利的剪刀（约5毫米），无论是在前肢背部或后肢腹部附近（ 图1）以上的微小切口。 </li><li>删除松散的头发，用钳子切断。如果你愿意，你可以刮胡子鼠标，然后才作出的切口。但是，一个较长的过程，我们发现，这是没有必要以来，如下所示，它不会改善愈合，减少感染，这都是非常罕见的。 </li><li> 2钳拉伸切口才能看到的LN。切口可能达到10毫米。号法律公告出现灰褐色或暗比周围脂肪。 </li><li>捏用一个镊子的LN上的筋膜（薄膜覆盖的脂肪和组织），轻轻拉，不破坏周围组织。 </li><li>将第二个钳尽可能underneath的LN。第一镊子，打破筋膜和删除号法律公告。如果您已成功收获的LN，染色的血液应该出现在其中的LN以前所在的位置。需要注意的是等渗溶液中的LN汇。这个简单的测试，让您验证您已提取的一种法律公告，而不是脂肪组织。 </li><li>确认没有毛发在伤口。 </li><li>坚持2部分皮肤（皮肤内），装订剪辑，关闭切口。我们使用应用镊子一些米​​歇尔夹。安装1或2个片段，切口的大小而定。大多数情况下，剪辑时脱落的组织愈合。 </li></ol> 3。术后护理<ol><li>笼底部添加一些湿的食物，手术后容易使老鼠能养活。 </li><li>手术后约6至8小时，检查，以确保他们没有经历过痛苦的小鼠。仔细观察他们的行为（姿势，步态，毛皮的外观与同类和相互作用）。如果小鼠的整体外观差，管理的丁丙诺啡第二剂量。由老鼠第二天应该恢复其正常的行为，否则，继续丁丙诺啡的管理。小鼠从未达到先前确定的，由动物保健理事会接受的终点。你可能想寻求咨询和帮助你照顾动物饲养箱技术员。 </li></ol> 4。淋巴结处理<ol><li>在实验室中，获得单细胞悬液，游离为您常​​用的程序的法律公告。我们LN和对磨砂幻灯片按常规获得1-2×10 6细胞每号法律公告。另外，我们准备RNA提取的LN。 </li><li>细胞染色为你平时的协议。你应该获得预期的LN流式细胞仪配置文件。 </li></ol> 5。代表结果例如前部和侧散射带够很多经手术或从牺牲的鼠标（ 图2）从麻醉鼠标收获淋巴结。在现场淋巴细胞门的形象和细胞的百分比相同，表明法律公告手术是一个合适的技术，收获细胞。 <img alt="图1" src="/files/ftp_upload/3444/3444fig1.jpg" /> 图1。腹股沟和鼠标臂LN的本地化。 A.腹股沟号法律公告位于略高于后肢，稍往肚子。 B.臂丛LN是位于前腿后面。 <img alt="图2" src="/files/ftp_upload/3444/3444fig2.jpg" /> LN的手术后得到图2。流量淋巴细胞流式细胞仪轮廓。前部和侧散射剖面显示从麻醉收获的淋巴结（一）经手术或从牺牲的鼠标（二）etized鼠标。淋巴结收获，分离和流式细胞仪分析。细胞在活的淋巴细胞门的百分比显示。

<ol>
	<li>Zehn, D., Lee, S. Y., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+but+curtailed+T-cell+response+to+very+low-affinity+antigen.">Complete but curtailed T-cell response to very low-affinity antigen.</a> Nature. 458, 211-214 (2009).</li><li>Vezys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8+T-cell+compartment+grows+in+size+with+immunological+experience.">Memory CD8 T-cell compartment grows in size with immunological experience.</a> Nature. 457, 196-199 (2009).</li><li>Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+persistence+of+memory+CD8(+)+T+cells+depend+on+T+cell+factor+1.">Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.</a> Immunity. 33, 229-240 (2010).</li><li>Wirth, T. C., Badovinac, V. P., Zhao, L., Dailey, M. O., Harty, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+central+memory+CD8+T+cells+is+independent+of+CD62L-mediated+trafficking+to+lymph+nodes.">Differentiation of central memory CD8 T cells is independent of CD62L-mediated trafficking to lymph nodes.</a> J. Immunol. 182, 6195-6206 (2009).</li><li>Rooke, R., Waltzinger, C., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+complementation+of+MHC+class+II+deficiency+by+intrathymic+delivery+of+recombinant+adenoviruses.">Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.</a> Immunity. 7, 123-134 (1997).</li><li>Witherden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-controllable+selection+of+CD4(+)+T+cells:+half-life+and+survival+signals+in+the+absence+of+major+histocompatibility+complex+class+II+molecules.">Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.</a> J. Exp. Med. 191, 355-364 (2000).</li><li>Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+TCR+does+a+T+cell+need.">How much TCR does a T cell need.</a> Immunity. 15, 71-82 (2001).</li><li>Leignadier, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+density+influences+CD8++memory+T+cell+differentiation.">Epitope density influences CD8+ memory T cell differentiation.</a> PLoS One. 5, e13740 (2010).</li><li>Leignadier, J., Hardy, M. P., Cloutier, M., Rooney, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T-lymphocyte+survival+does+not+require+T-cell+receptor+expression.">Memory T-lymphocyte survival does not require T-cell receptor expression.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20440-20445 (2008).</li><li>Obst, R., van Santen, H. M., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+persistence+is+required+throughout+the+expansion+phase+of+a+CD4(+)+T+cell+response.">Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response.</a> J. Exp. Med. 201, 1555-1565 (2005).</li><li>Bassett, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T-cell+expansion+and+maintenance+after+recombinant+adenovirus+immunization+rely+upon+cooperation+between+hematopoietic+and+nonhematopoietic+antigen-presenting+cells.">CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.</a> Blood. 117, 1146-1155 (2011).</li><li>Lacombe, M. H., Hardy, M. P., Rooney, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+receptor+expression+levels+do+not+identify+CD8++memory+T+lymphocyte+precursors+following+peptide+immunization.">IL-7 receptor expression levels do not identify CD8+ memory T lymphocyte precursors following peptide immunization.</a> J. Immunol. 175, 4400-4407 (2005).</li><li>Leignadier, J., Rooney, J., Daudelin, J. F., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lowering+TCR+expression+on+naive+CD8++T+cells+does+not+affect+memory+T-cell+differentiation.">Lowering TCR expression on naive CD8+ T cells does not affect memory T-cell differentiation.</a> Immunol. Cell. Biol. 89, 322-325 (2011).</li></ol>

没有利益冲突的声明。

我们已经描述了LN的手术协议，它可以适用于许多实验系统。虽然非常有益的免疫反应或免疫细胞的平衡，这种技术具有一些限制。首先，只有四个LNS可以收获。二，免疫反应的研究必须是系统性的，或发生在腹股沟浅淋巴结或臂丛（皮肤排出）。最后，收获细胞数号法律公告搬迁的质量，技术的限制，这可能有助于实验变异时需要绝对细胞数的影响。然而，对靶细胞的百分比，对自己，可以提?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>格雷夫精细模式高级钳</td><td>哈佛仪器</td><td> 52-2144 </td><td>强烈弯曲，10厘米（4英寸），0.8毫米尖</td></tr><tr><td>米歇尔剪辑应用和删除钳</td><td>哈佛仪器</td><td> 52-3779 </td><td> 12.5厘米（5） </td></tr><tr><td>米歇尔剪辑100 </td><td>哈佛仪器</td><td> 52-3746 </td><td> 7.5-1.75毫米</td></tr><tr><td> temgesic（盐酸丁丙诺啡） </td><td>先灵葆雅</td><td></td><td></td></tr><tr><td> vetalar（盐酸氯胺酮） </td><td> boniche动物保健</td><td></td><td>根据DIN：01989529 </td></tr><tr><td> rompun（二甲苯胺噻嗪） </td><td>拜耳</td> &lt;TD&gt; </td><td>根据DIN：02169592 </td></tr><tr><td>异氟醚</td><td>雅培</td><td></td><td>根据DIN：02032384 </td></tr><tr><td> hypotears眼膏</td><td>诺华Ophtalmics </td><td></td><td>根据DIN：02133288 </td></tr><tr><td> baxedin（Clorhexidine葡萄糖2％，异丙醇70％） </td><td>欧米茄</td><td> L0000017 </td><td>根据DIN：02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

在免疫学领域，了解进展对疫苗，感染或肿瘤的免疫反应，反应之后往往随着时间的推移。同样，淋巴细胞稳态的研究需要时间课程实验。这些研究是在相同的鼠标理想，以减少实验的变异，以及使用的老鼠数量。抽血时间课程实验允许的性能，但它只能提供约外周血淋巴细胞的信息，并提供数量有限的细胞1-4。由于通过人体循环和居住在淋巴结中的淋巴细胞有不同的特性，重要的是要检查这两个地方。淋巴结顺序切除手术提供了一个独特的机会，后续的免疫反应或免疫细胞膨胀，随着时间的推移相同的鼠标。此外，这种技术的产生，这是足够的PE 1-2X10 6个淋巴结细胞之间rform表型特征和/或功能分析。顺序淋巴结手术或淋巴结清扫已成功地应用于我们和其他国家5-11。在这里，我们描述如何可以通过一个小切口，在皮肤一个anesthetised鼠标删除臂丛和腹股沟淋巴结肿大。由于手术是肤浅的，并迅速完​​成，鼠标恢复速度非常快，愈合好，不会遇到过多的疼痛。每隔一天，有可能收获一个或两个淋巴结允许时间课程实验。因此，该技术是适合学习节点居住淋巴细胞淋巴结的特点，随着时间的推移。这种方法适用于各种实验设计，我们相信，许多实验室将受益于执行顺序淋巴结手术。

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

遵循多时间内相同的鼠标免疫反应的进展，淋巴结可以依次通过手术切除。在这里，我们描述了这种技术可以如何执行。

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 ...

Research

JoVE Journal

Immunology and Infection

42.0K 次观看.  Maisonneuve-Rosemont Hospital Research Center. 遵循多时间内相同的鼠标免疫反应的进展，淋巴结可以依次通过手术切除。在这里，我们描述了这种技术可以如何执行。

视频: Murine Superficial Lymph Node Surgery

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 ...

科研

免疫与感染

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

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

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

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

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 ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...