This work was supported by funding received from the National Institutes of Health (1R01AI083269).

鼠研究批准了北卡罗来纳教堂山大学的机构动物护理和使用委员会（IACUC）和所有的动物护理是按照IACUC的指导方针。 1.准备新生儿和成人Thymi的<ol><li>准备所有安乐死供体小鼠之前，试剂和设备。 </li><li>消毒手术器械通过高压灭菌或其他合适的方法。所有的外科手术必须在层流罩保持无菌条件和避免污染进行。组装需要从供体小鼠提取thymi的工具。 </li><li>填充60mm培养皿用无菌的1×PBS（pH7.4）中，并放置在冰上的罩内。这将被用于简单地存储thymi从供体小鼠移植前切除。 </li><li>按道德准则，通过CO新生儿安乐死供体小鼠斩首和成年供体小鼠2窒息颈椎脱位。 </li><li>当r胸腺emoval:莱鼠标在背卧位在无菌吸水纸巾喷洒之前做一个切口70％的乙醇。 </li><li>通过皮肤制备中线切口暴露腹腔和胸腔。折叠的皮肤在胸部和前肢以显示胸腔。 </li><li>使通过隔膜和肋骨两侧切口，显露上纵隔和前胸腔。胸腺应该是可见的两个白色的凸起立即上方和相邻的心脏。 </li><li>除了捉弄周围用细镊子胸腺结缔组织，使某些不破坏胶囊。而憋尿用钳子胸腔，使用第二对镊子通过定位弯钳器官下方，垂直提拉提取胸腺的两叶。这可以使用解剖范围为从新生小鼠提取thymi来完成。 </li><li>放置在胸腺60毫米双含有SH在冰上并分离胸腺裂片无菌的1×PBS（pH7.4）中，通过所述连通峡部切割。从胸腺裂片使某些不损坏胶囊除去任何碎片，并切断胸腺成用于移植部分的适当数量。 </li><li>不要操纵新生小鼠获得的thymi。 注:用于最多两个受体小鼠（每个收件人1叶）中。 </li><li>移植从成年小鼠中得到的thymi分为最多4-6受体小鼠。使用一对镊子，小心地抓住一名成人胸腺叶， 如图1，切到胸腺用手术剪刀三个相等的部分。 </li><li>重复步骤1.3-1.4为每个供体小鼠。为了限制thymi暴露的时间，准备1施主胸腺的时间。 </li></ol> 2.植入胸腺肾囊下<ol><li>装配在表1中列出的预先消毒设备，适当的无菌技术应的过程，以防止暴露移植受者对污染的工具或试剂的过程中被利用。 </li><li>移植前，权衡和标记每个收件人鼠标。 </li><li>设置解剖显微镜和麻醉电路在层流罩。 </li><li>使用电动剃须刀，剃接受者小鼠的左侧，并确保没有头发仍然围绕用于使切口的区域。 </li><li>打开异氟烷蒸发器和麻醉使用的1-2％之间的剂量鼠标。通过检查以下脚趾捏缺乏反射开始手术前确定适当的麻醉。 </li><li>之后，鼠标正常麻醉，适用兽医药膏鼠​​标以防止干燥和准备鼠标移植如下的眼睛: </li><li>定位在解剖显微镜下鼠标在右侧卧位，使剃侧朝上。 Starti纳克在手术区的中心，使用一次性移液管的70％乙醇，随后碘伏（优碘）分配在一个圆周运动。重复乙醇/优碘治疗3次之前做一个切口。 </li><li>使用解剖剪刀做一个1-2厘米的切口侧翼肾脏正上方。 </li><li>使用镊子中等掌握邻近肾脏结缔组织和轻轻抬起肾使得其位于上盖的肌肉。为了保持肾脏暴露和地方在肌肉组织，插入介质镊子一只手臂肾下，要特别注意不要破坏肾髂骨。 </li><li>为了防止组织脱水，在每个步骤中，直至肾和肾囊操纵完成灌溉用无菌的1×PBS（pH7.4）中的肾。 </li><li>用细镊子夹住并提起肾脏最远端的肾上腺它从肾脏分离的边缘附近的胶囊。 </li><li>使用18号针，使一个切口足够大以插入准备好的片的供体胸腺（ 图2A）。使一定保持囊切口尽可能小，以防止移植物的错位随着时间的推移。 </li><li>而从肾脏保持胶囊拉远，使用第二对细镊子的插入胶囊下方的移植物和胸腺从囊切口尽可能（ 图2B）推远向前。 </li><li>从肾脏下方取出钳，并通过切口轻轻返回肾脏回原位。 </li><li>缝合腹膜壁和应用一旦关闭优碘关闭肌肉。缝合完成后，应用伤口夹关闭真皮并应用优碘到周边地区。 </li><li>返回鼠标已使用热灯加热笼子。 </li><li>监测每个术后鼠标，直到它恢复全部的意识和行动，也不要将鼠塔吨有没有恢复麻醉下与其他动物的笼子。 </li><li>根据需要治疗的小鼠手术后的疼痛管理药物。提供小鼠手术后止痛药如对乙酰氨基酚在饮用水以1.6毫克/毫升的浓度。 </li><li>重复步骤2.3-2.9为每个收件人鼠标。 </li><li>确保小鼠恢复正常活动中1小时后的手术。监视手术后的重量，运动技能，以及各小鼠的饮用和摄食活动，以确定所产生的移植过程的结果的并发症。 注:手术后体重减轻，相比手术前的重量，以及改变流动性或没有吃或喝可以指示并发症。 </li><li>监视外周血T细胞重建的出血的小鼠经尾缺口随后根据制造商的说明书使用Lympholyte细胞分离介质的淋巴细胞的分离。实现T细胞characteriza灰通过染色的淋巴细胞与特异于T细胞标记CD3，CD4，和CD8，以及一个活死鉴别荧光标记的抗体，例如荧光激活琥珀酰亚胺基酯8。 如图3分析可以通过选通上单峰，活细胞，和CD3阳性细胞来完成。 </li></ol>

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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

胸腺是在T细胞发育的关键事件发生1器官。驻地胸腺细胞，在T细胞受体（TCR）α和β基因的重排，进行一系列与在胸腺2的皮质和髓质区lymphostromal和抗原呈递细胞（APC）的相互作用。胸腺阳性选择由皮质胸腺上皮细胞（TEC）介导，以产生胸腺细胞识别的抗原肽在宿主主要组织相容性复合体（MHC）I类和II类分子2-3的上下文中。随后胸腺阴性选择嗣继承吹扫自身反应的胸腺细胞，通过与髓TEC或树突细胞（DC）从自身蛋白质结合的MHC I类和II类分子3推导出本发明的肽的相互作用来驱动。这些处理的最终结果是建立成熟的CD4 +和CD8 + T细胞能够对广谱响应一池外来抗原的同时表现出最小的反应性的自身蛋白质4。 胸腺选择事件的效率是由许多因素，包括胸腺成熟，髓和皮质TEC，胸腺DC亚群组成的频率，和胸腺前体3的源的影响。值得注意的是，异常胸腺选择可导致自身免疫性5或免疫缺陷的病状，从受损的阴性或阳性选择，它们分别出现。的分子事件调节胸腺选择，然而，却知之甚少。 体外方法，如reaggregate胸腺器官培养（RTOC）6，已被证明是用于分析与胸腺选择相关联的基本事件是有用的，但不能完全概括的正在进行的动态在体内的活动。这样一来，这个胸腺移植为基础的方式成立，以便更好地研究胸腺细胞评选活动在体内 7 </s向上&gt;。 本协议描述移栽从新生和成年供体小鼠thymi到免疫缺陷SCID受体小鼠。这种技术允许调节阳性和阴性胸腺选择，以及各种T细胞亚群的个体发育过程中胸腺输出机制的研究。最近，这种方法已被用于证明胸腺选择的效率出生的小鼠，导致自身反应性T细胞的增加发展后早期的限制，并降低T细胞剧目特异于外来抗原7。

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			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
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		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
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		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
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isolation and transplantation of different aged murine thymic grafts.

调节胸腺选择的有效性的机制仍然不明确。这里介绍的方法允许在体内特异于自我和外来抗原的T细胞的发展和选择的分析。该方法需要来自各种老年小鼠免疫缺陷SCID收件人胸腺移植物的植入。在一段相对较短的时间的收件人完全重构与来自植入胸腺移植物衍生的T细胞。只有播种的胸腺细胞在胸腺在隔离的时间进行选择，并发展成为成熟的T细胞。这样，改变在移植的T细胞的年龄依赖性胸腺事件的函数的性质和特异性可以进行评估。虽然技术专长需要成功胸腺移植，这种方法提供了以研究在体内的广泛的病理是由于异常胸腺功能和/或homeostas或结果的一个独特的战略是。

这个过程的成功依赖于最小的手术创伤，以及肾囊下的移植物的准确定位。胸腺移植物应该被切割，以确保适当大小胸腺部分用于随后的移植， 如图1。按照图1中的示意图，从新生或成年小鼠thymi可用于成功包膜下移植具有一致和可重复的T细胞植入的结果。如所提到的，肾囊下的移植物的适当的定位是在保持长期存活的移植物，功能，和血管重要。正如在图2...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

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Immunology and Infection

13.7K 次观看.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

视频: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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 ...

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 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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

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 ...