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。

<table><tbody><tr><td>DMEM</td><td>LifeTechnologies-Gibco</td><td>41965-039</td><td></td></tr><tr><td>FCS</td><td></td><td></td><td>Final concentration 2%</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>499609</td><td>Final concentration 1.2 mM</td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td></td><td>&gt;160 units per mg dry weight, use at final concentration of 1 mg/ml</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>use at 3.5 mg/ml</td></tr><tr><td>DNAse I</td><td>Roche&amp;nbsp;</td><td>11284932001</td><td>use at 40 &amp;micro;g/ml</td></tr><tr><td>Stiring magnets</td><td>FAUST</td><td></td><td>5 mm long-2 mm &amp;oslash;</td></tr><tr><td>Polystyrene round bottom tubes 5 ml</td><td>Falcon-BD Bioscience</td><td></td><td></td></tr><tr><td>Magnetic stirrer with heating function</td><td>IKA-RCT-standard</td><td>9720250</td><td></td></tr><tr><td>Petri dishes 100 mm, sterile</td><td>TPP</td><td>6223201</td><td></td></tr><tr><td>25 G needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>anti mouse CD45 Ab</td><td>Biolegend</td><td></td><td>Clone 30-F11</td></tr><tr><td>anti mouse CD11c Ab</td><td>Biolegend</td><td></td><td>Clone N418</td></tr><tr><td>anti mouse Podoplanin Ab</td><td>Biolegend</td><td></td><td>Clone 8.1.1</td></tr><tr><td>anti mouse CD31 Ab</td><td>Biolegend</td><td></td><td>Clone MEC13.3</td></tr><tr><td>&lt;br/&gt; Eppendorf Xplorer plus, Multichannel</td><td>Eppendorf</td><td>4861 000.821/830</td><td>1.250 &amp;micro;l max. volume</td></tr><tr><td>anti mouse CD140a</td><td>Biolegend</td><td></td><td>Clone APA5</td></tr><tr><td>anti mouse CD80</td><td>Biolegend</td><td></td><td>Clone 16-10A1</td></tr><tr><td>anti mouse CD40</td><td>Biolegend</td><td></td><td>Clone 1C10</td></tr><tr><td>anti mouse I-Ab</td><td>Biolegend</td><td></td><td>Clone AF6-120.1</td></tr><tr><td>anti mouse CD274 (PD-L1)</td><td>Biolegend</td><td></td><td>Clone 10F.9G2</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td><td>Invitrogen</td><td>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

Research

JoVE Journal

Immunology and Infection

31.2K 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

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

免疫与感染

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

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

小鼠小肠淋巴细胞的分离和流式细胞表征

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

Isolation and Activation of Murine Lymphocytes

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

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

分离及小鼠淋巴细胞的激活

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

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

小鼠小肠免疫系统淋巴细胞的分离

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

优化从胸腺和骨髓中分离基质细胞

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

鼠淋巴结基质细胞的分离

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