The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

在这个视频发布和协议，所有动物的程序按照批准的州局巴塞尔城市，瑞士动物的协议进行的。 1，淋巴结制备及消化<ol><li>在烧杯中，以37℃的磁力搅拌器与加热板预热水。 </li><li>制备基本培养基如下:DMEM培养基（不含丙酮酸）补充有2％FCS，1.2mM的氯化钙和青霉素/链霉素（100单位的青霉素，100微克链霉素）。 </li><li>在使用前消毒所有的解剖工具。 </li><li>安乐死每CO 2窒息淋巴结供体小鼠和在无菌条件下解剖淋巴结。不要解剖周围的脂肪。注:此协议是周围皮肤引流淋巴结（腹股沟，臂丛，腋）进行了优化。 </li><li>放置淋巴结中含2ml冰冷的基本培养基的无菌培养皿中。 </li><li>扰乱淋巴结CApsule使用两个地下25针固定在1ml注射器。 </li><li>传输中断淋巴结组织中含有补充了1毫克/毫升胶原酶IV和40微克/毫升DNA酶一750μl的基本培养基5ml的聚丙烯圆底试管</li><li>每管加一无菌磁力搅拌器。 </li><li>在37℃的烧杯放置管预热水搅拌管以缓慢的速度（1转/秒）30分钟。 </li><li>从磁力搅拌器与加热板拆下管子，让淋巴结片段定居。 </li><li>小心取出富含"非基质细胞"上清。注:如果T，B细胞，树突细胞和CD45的分析- GP38 - CD31 -预见，保存"非基质细胞"部分。 </li><li>洗涤剩余淋巴结组织一次与750微升的基本媒介。注:此步骤仅当工作与免疫能力的小鼠必要的。 </li><li>让淋巴结片段定居。 </li><li>取出非基质细胞漂浮分数。 </li><li>添加到淋巴结片段750微升的基本培养基中添加3.5毫克/毫升胶原酶D和40微克/毫升的DNase一</li><li>地方管早在含37℃烧杯预热水。 </li><li>消化淋巴结组织为5分钟，同时慢慢搅拌。 </li><li>集计淋巴结组织碎片通过移液和混合700微升，10个循环的最大速度使用自动化多通道移液器。注意:这会破坏淋巴结组织，以改善消化。 </li><li>将管子放回烧杯中37℃预热的水。 </li><li>摘要淋巴结组织碎片的同时，慢慢搅拌另外10分钟。 </li><li>分解淋巴结组织碎片通过移液和使用自动化多通道移液器混合为99个循环的最大速度。 </li><li>加入7.5微升0.5M的EDTA，确保维修单细胞悬液。 </li><li>由管集计淋巴结组织碎片拟合和使用自动化多通道移液器混合为99个循环的最大速度。 </li><li>加入750微升碱性介质中，并通过70微米尼龙网传递细胞。 </li><li>离心细胞悬浮液5分钟，在1500 XG，4℃。 </li><li>基质细胞现在可以被用于进一步的分析。 </li></ol> 2，染色淋巴结基质细胞<ol><li>使用抗CD45，抗平足（GP38），抗CD31抗体的结合，成功地承认真相与和解委员会，LEC，BEC和DN细胞。 </li><li>孵化用活/死跟踪器，抗CD45-FITC（1:200）的消化淋巴结组织，抗GP38-PE（1:200），抗CD31-APC（1:200）在100μlHBSS含2 ％FCS的为至少20分钟，4℃下，在黑暗中。 </li><li>洗含2％FCS的细胞中加入500升的HBSS </li><li>离心细胞悬浮液3分钟，在1500 XG，4℃。 </li><li>重新悬浮在含有2％FCS的100微升HBSS中。 </li><li>运行染色的细胞在流式细胞仪equipp版具有以下光学系统:激发源与多达三个激光器:蓝色（488纳米，风冷，20毫瓦固态），红色（633纳米，17毫瓦的氦氖），紫（405纳米，30毫瓦固态） 。 </li><li>门CD45 -细胞排除造血细胞。 </li><li>门汗衫（FSC-W）和活细胞（活/死追踪器）排除双峰和死细胞。 </li><li>情节GP38 与 CD31的形象化TRC（GP38 + CD31 - ），LEC（GP38 + CD31 +），BEC（GP38 - CD31 +）和DN（GP38 - CD31 - ）细胞（参见图1）。 </li></ol>

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The authors declare they have no competing financial interests.

淋巴结基质细胞的研究最近成为研究的热点，由于两个出版消化协议6,13的发展。这两种协议是足够获得单个淋巴结基质细胞，但不同之处在于使用消化酶和消化的时间。自基质细胞和其表面标志物是酶消化和机械应力敏感，一个优化的协议是必须的。 可行的淋巴结基质细胞从新鲜解剖淋巴结的隔离是为了进行表型和功能的分析的第一步。因此，在限定的酶浓度要仔?...

淋巴结有专门的隔间，其中对外国和自身抗原的适应性免疫应答发起和协调。这里介绍的过程描述很短的酶消化结合机械式自动移液获得淋巴结单细胞悬液，并获得可行的淋巴结基质细胞的维持几个分子的表面表达。 淋巴结基质细胞形成淋巴结的支架，履行三大职能:第一，他们筛选体液样品抗原，病原体及其病原相关分子模式（的PAMP），以及细胞因子与危险相关分子模式（D-AMPS）目前在体内。第二，它们吸引和指示的抗原呈递细胞（APC）和淋巴细胞相互作用，并启动适应性免疫应答;第三，他们为淋巴细胞1的稳态和分化的结构性环境-3。在炎症过程中淋巴结基质细胞产生的生长因子，细胞因子和趋化因子，适应肿胀从而组织树突状细胞（DC）之间的相互作用，T，和B细胞。免疫反应的编排是唯一可能的，因为由不同的基质细胞群体形成的复合结构的体系结构。 淋巴结基质细胞是CD45阴性细胞中，并且可以通过CD31的表达或GP38在成纤维细胞和内皮细胞1-6相区别。 GP38 + CD31 -定义T区网状细胞（TRC，也被称为FRC:成纤维网状细胞），GP38 + CD31 +限定淋巴管内皮细胞（LEC），GP38 - CD31 +限定血液内皮细胞（BEC）。此外，该亚群的特征显示其他淋巴结基质细胞的存在。事实上，小周细胞样细胞群进行了表征内GP38 - CD31 -人口7。因此，在分离过程中的适应是有利的，用于鉴定不同淋巴结基质细胞的功能特性和特征。 的淋巴结基质细胞消化之前开发协议淋巴结基质细胞的研究是利用组织切片和显微镜不限于原位观测。然而，结构和功能研究表明，淋巴结基质细胞的重要特征。淋巴结基质细胞与平足，胶原和细胞外基质（ECM）蛋白结合以形成所谓的管道系统中的复杂的三维结构，其输送淋巴和相关联的低分子量蛋白质的淋巴结的囊下窦的高内皮微静脉中的T细胞区8。 DC是在与基质细胞紧密接触，并且可以观察到突出到管状CONduit结构来样液和检测抗原8。淋巴结基质细胞（储税券和淋巴管内皮细胞）与DC的相互作用是由趋化因子CCL21和CCL19 9,10释放和呈现介导的。 CCL19和CCL21通过CCR7受体促进树突状细胞和T细胞迁移至淋巴结的T细胞区4,11确认。尽管使用类似的趋化因子，树突状细胞和T细胞有不同的迁徙路线进入淋巴结12。后来，使用淋巴结和纯淋巴结基质细胞分离的酶消化，功能研究，对不同淋巴结基质细胞的作用和其与DC和T / B细胞6,13相互作用的能力进行的。首先，IFN-γ的产生的效应T细胞和淋巴结基质细胞之间的交调失真引起的生产所示以抑制T细胞应答和增殖中的次级淋巴器官14-16的代谢物的一氧化氮。二，淋巴Ñ赋基质细胞已报告，以支持监管DC亚群的分化通过产生IL-10的17的，并通过产生IL-7 6,18的调节幼稚T细胞稳态。三，淋巴结基质细胞Toll样受体的表达表明，基质细胞很容易受到信号从组织损伤时释放的感染或自身分子而得。的确，淋巴结基质细胞与TLR3聚配体的治疗（I:C）诱导主要组织相容性复合体I类表达和共同抑制分子PD-L1的表达上调的适度上调，但不是共刺激分子，从而导致外周组织的急剧变化抗原表达19。几个小组已经表明淋巴结基质细胞表达外周组织中的抗原并诱导自身反应性T细胞19,21-27的耐受性。因此，了解淋巴结基质细胞和其它迁移和再之间的相互作用目前主席淋巴结细胞，将有助于找到新的目标分子，以允许在炎症期间激活或抑制免疫应答。因此，淋巴结的出版酶的分离的实施是必要的。 先前公布的协议使用胶原酶类酶消化具有低机械应力6,19,20的不同组合。然而，长期的孵化与消化酶或消化酶的不同组合，可能会降低分析的激活状态，并确定新的淋巴结基质细胞所需的各种表面分子。根据不同的基质细胞的分析的类型，链路协议或弗莱彻协议可能是更适合的。在所描述的过程中，稍短酶消化结合了自动机械解聚作用，以减少可行淋巴结基质细胞的表面标记物的降解。此过程使高度可重复的分离和区分淋巴结基质细胞群与低变异性和超过95％的存活率。将新鲜分离的淋巴结基质细胞可直接用于表面标志物表达，蛋白质分析和转录的研究，以及建立基质细胞系在体外进行功能测定。

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			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

本协议是出版Link 等人 ，2007年6具有更短的消化时间（45分钟，最大）由于机械解聚与自动化多通道移液管的变形消化协议。此外，该过程是更加标准化，最大限度地减少了对不同淋巴结的基质细胞表面标记物的降解，并且允许多于一个样品的处理在同一时间。 胶原酶IV和胶原酶D IN链接协议6和目前的协议或胶原酶P和中性蛋白酶到-Fletchers协议13?...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

31.1K 次观看.  University of Basel and University Hospital Basel. 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. 

视频: Isolation of Murine Lymph Node Stromal Cells

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 ...

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 ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

小鼠牙龈细胞的分离，处理和分析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

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, ...