这项工作是由卫生部资助R01 AI088209（到KAH）国家机构的支持。我们也感谢明尼苏达流式细胞资源的大学。

在这项研究中，成人（6 - 8周）雌性C57BL / 6小鼠。小鼠是从国立癌症研究所购买和无特定病原体的条件下维持。明尼苏达机构动物护理和使用委员会大学（IACUC）批准的所有动物实验。 1，准备工具和缓冲区<ol><li>制备的酶溶液（RPMI1640培养基用0.05％[w / v的] LIBERASE TH和DNA酶I的100单位/毫升的），白蛋白富含缓冲液（1X PBS中的[Ca 2 + / Mg 2 +的 -free]用0.5％牛血清白蛋白和2mM乙二胺四乙酸[EDTA]），FACS缓冲液（1X PBS中的[Ca 2 + / Mg 2 +的 -free]含有1％胎牛血清[FBS]和0.02％NaN 3的），FACS分选缓冲液（1X PBS有10％FBS的[Ca 2 + / Mg 2 +的 -free]含有2％FBS）中，并RP10培养基（RPMI-1640培养基中提供）。 </li><li>准备10淘洗板。制备40毫升涂层溶液（0.01毫克/毫升ģ燕麦抗大鼠IgG的0.1M的NaHCO 3缓冲液），并加入4毫升涂布液到每100×15毫米板，漩涡和孵育在4℃CO / N。倒掉抗体溶液冲洗，用5毫升的1X的PBS中的2倍，板，旋流大力冲洗之间。存储板准备在4℃。 </li></ol>胸腺组织2丰收<ol><li>安乐死鼠标在CO 2室。把动物在其上的夹层垫回，PIN脚在垫子上，并用70％乙醇喷洒湿了的皮草。 </li><li>作中线切开穿过皮肤，从尿道口上方直线上升到下颚用剪刀开始和向下延伸的切口，以两侧的膝盖。切口看起来像一个倒置的“Y”。拉扯皮肤松弛背部的两侧，并将其引脚连接到夹层垫。 </li><li>从剑突刺穿隔膜，切断两侧的肋高达约锁骨上，然后向上抬起与肋镊子，针到夹层垫。胸腺只是肋骨下，看起来像两条细细的白瓣覆盖心脏（ 图1）。断开结缔组织包围胸腺，轻轻一拉，取下一对胸腺叶与弧形锯齿钳。地方胸腺裂片入含有5ml RPMI-1640 6孔板。 </li></ol> 3，准备胸腺基质细胞<ol><li>修剪任何周围脂肪和结缔组织和胸腺瓣转移到含有5毫升新制备的酶溶液中的6孔板的一个新井。作小切口在胸腺裂片细剪刀，孵育该板在37℃下进行20分钟。 </li><li>轻轻吸取消化胸腺组织上下2 - 3用5毫升吸管进行组织分离时间。收集在50ml管中含有10毫升冷的白蛋白富缓冲器中和酶的上清液部分。保持在冰上的收集管。添加2.5毫升酶溶液的剩余组织孵育该板在37℃下进行15分钟。 </li><li>用3毫升注射器和18G的针头打散聚集进行轻柔的机械搅拌。池上清液到收集管中。添加2.5毫升酶溶液的剩余组织孵育该板在37℃下进行15分钟。 </li><li>温柔的重复机械搅拌了3毫升注射器及地下25针，孵化盘增加5 - 10分钟。 </li><li>完全消化后，凝聚上清液进入收集管中。离心机在400 XG，4℃，8分钟，收集细胞的收集管。吸出的液体和悬浮沉淀的细胞在10ml白蛋白富含缓冲和过滤到100毫米筛网的样品。计数使用血球细胞。然后进行流式细胞术分析（第4节），或富集的TEC（第5章）。 </li></ol> 4，肾小管上皮鉴定通过流式细胞仪</p&gt; <ol><li>制备的细胞以每100μl的FACS缓冲液用于染色4×10 6个细胞的最终浓度在96孔圆底测试板，并保持该板在冰上。加入20μlFc的块溶液（抗CD16 / CD32抗体在10微克在FACS缓冲液/毫升）至细胞中，并孵育细胞，在冰上10分钟。旋转该板在400 XG，4℃，3分钟。轻弹板面朝下成片丢弃井中的液体。 </li><li>暂停沉淀的细胞中加入100μl抗体混合物（抗-CD45，-EpCAM，β-MHC II类和-Ly51抗体，和UEA-1凝集素标记不同的荧光染料，在制造商推荐的或在FACS缓冲液测试每种抗体的最佳浓度），并孵育细胞在冰上20分钟，在黑暗中。为补偿，染色1×10 6个细胞与每个荧光染料。 </li><li>添加100μl的FACS缓冲液到每个孔中，并旋转该板在400 XG，4℃，3分钟。重复该洗涤步骤1时间，然后重悬细胞于100μlFACS缓冲液。将细胞转移到一个12×15毫米圆底流式细胞仪套管内填充300微升FACS缓冲液。获得的流量采样仪和使用流式细胞仪分析软件分析数据。 TEC门控策略示于图2。 </li></ol> 5，丰富的TECs通过平移<ol><li>降速的胸腺细胞（在第​​3节中制备），在400 XG，4℃5分钟。吸出的液体和悬浮沉淀的细胞在FACS分选缓冲液以每毫升2×10 8个细胞在15ml或50ml锥形管中的浓度。添加抗CD90.2抗体（克隆30-H12）（或抗CD45），在细胞悬浮液中的2.5微克/毫升的最终浓度，并轻轻孵育细胞在旋转混合器中，在4℃下进行30分钟。以下的温育，洗涤用5 - 10倍体积RP10培养基并离心管中在400 XG，4℃5分钟。 </li><li>吸液和悬浮沉淀细胞以1×10 7每毫升浓度RP10媒体。加入5 ml的细胞悬液，以每涂覆淘洗板中（在步骤1.3中制备的），涡旋并温育30分钟，在室温。大力漩涡;上清转移至锥形管。冲洗该板用RP10培养基两次，集中上清液进入收集管中。 </li><li>离心收集管中，在400 XG，4℃5分钟。吸液重悬细胞沉淀在5毫升RP10媒介。添加细胞悬浮液到一个新的淘洗板中，漩涡及温育30分钟，在室温。收集细胞冲洗板，集中成一个锥形管。一旦肾小管上皮富集，降速细胞在400 XG，4℃，5分钟，细胞悬浮在5毫升流式细胞仪分选缓冲。 </li></ol>通过细胞分选6，净化的TECs <ol><li>计数使用血球细胞和制备细胞悬浮液在4×10 7个细胞的FACS分选缓冲液中的浓度。添加抗体DEScribed在4.4节到细胞溶液并轻轻孵育细胞在旋转混合器中，在4℃下进行30分钟。以下染色，洗涤并悬浮细胞，以每毫升的FACS分选缓冲液10×10 6个细胞的浓度。 </li><li>排序通过100微米的喷嘴TEC的子集，利用荧光激活细胞分选。排序细胞被收集在30％（V / V）胎牛血清的RPMI-1640）。一旦细胞分类完成后，离心细胞，在400 XG，4℃，5分钟，并用于下游分析做准备。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoepithelial+interactions+in+the+mouse+thymus:+phenotypic+and+kinetic+studies+on+thymic+nurse+cells.">Lymphoepithelial interactions in the mouse thymus: phenotypic and kinetic studies on thymic nurse cells.</a> J Immuno. 128, 2287-2294 (1982).</li><li>Nakagawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+nurse+cells+provide+microenvironment+for+secondary+T+cell+receptor+alpha+rearrangement+in+cortical+thymocytes.">Thymic nurse cells provide microenvironment for secondary T cell receptor alpha rearrangement in cortical thymocytes.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 20572-20577 (2012).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J. Immunol. Method. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of immunological method. 385, 23-34 (2012).</li></ol>

作者宣称，他们没有竞争的经济利益。

在协议中，关键步骤是准备胸腺基质细胞（第3节）和肾小管上皮的富集（第4节）。强烈建议新鲜酶溶液，每次准备和组织，尽快治疗。对于池thymi，优化的酶溶液的体积取决于thymi的数量是必需的。如果任何组织残留物的处理之后左，加入更多的酶溶液和延长温育时间可以提高细胞产量。中富集的TECs，完成收集上清液，并重复在第5.3节中概述将降低信元丢失的板冲洗。 1980?...

早在T细胞发育，骨髓造血干细胞衍生的多潜能祖细胞被募集到胸腺的皮质，经受承诺T系和成为T细胞的前体1。在皮层，T细胞前体的CD4和CD8双阴性（DN）胸腺扩大和分化为不成熟的CD4和CD8双阳性（DP）的胸腺细胞，祖细胞形成具有高度可变的T细胞受体1的大型游泳池。仅DP细胞的选择MHC限制的子集将成为CD4或CD8单一阳性（SP）的胸腺细胞，迁移至胸腺髓质，并分化为功能性主管成熟的T细胞，这被称为正选择2的事件6。与此相反，自身反应性胸腺细胞克隆进行阴性选择，并通过细胞凋亡被删除，转换为调节性T细胞的自身耐受，或改行上皮内淋巴细胞的目的是尚不清楚-3,7-10。 在胸腺，胸腺基质细胞形成了独特的微环境提供这些不同的T细胞发育命运5,11,12信号。胸腺基质细胞组成的胸腺上皮细胞（的TECs） -包括皮层的TECs（cTECs）和延髓的TECs（mTECs），树突细胞，巨噬细胞，成纤维细胞，内皮细胞，神经脊衍生的周细胞等间充质细胞13-15。在这些中，的TECs是在T细胞发育1,2,16,17的各阶段的关键。然而，缺乏一个可靠的方法来隔离的TEC阻碍了它们的功能16一个全面的了解。特别是，cTECs，形成了周围的皮质祖细胞的三维网络，是必不可少的阳性选择13,18,19，其原因尚不清楚。早期的研究提供线索的TEC的异质性和作用，主要依靠形态学和组织学的工具13 </suP&gt;。最近TEC子集的独特作用是通过基因的方法在小鼠模型中12,20解决。一个强大的和可重复的方式来隔离的TEC是根本实现TEC的子集，对TEC的功能，定量和定性评估，并澄清机制，公正的鉴定cTECs如何支持正选择。 由于肾小管上皮在胸腺的稀有和紧张的互动，他们在完整的器官形成，肾小管上皮的分离一直是具有挑战性的。这里所描述的协议是基于先前的发现，目前可获得的试剂，技术和胸腺结构和基质组合物的知识。大约20年前，有几个程序被上报分解胸腺组织21-27，在不同的酶在消化过程中使用，包括胰蛋白酶，胶原酶和分散酶。 Gray 等人 。相比于他们的precedure 28的酶，并报告改进的甲基OD与酶鸡尾酒29，成为一个多步骤的消化广泛应用于20,30。然而，这种方法涉及到一个长的准备时间和复杂的消化步骤和结果变量最终细胞数和的TECs的比例，即使在同一小鼠队列29,30。几年前，LIBERASE研究级酶，含有高度纯化的胶原酶和中性蛋白酶启动在胸腺组织离解30中使用。在这里，我们描述了一个LIBERASE消化为基础的协议，具有优化的机械分离过程，能产生大量的可行的TEC的小鼠胸腺组织。 。 为丰富分离的间质细胞，以前的研究中使用任何浓度梯度或磁珠分离21,29。然而，两种方法都导致严重的损失胸腺基质细胞，cTECs 29的特别是某些人口的人口。细胞毒性消除和平移技术<s达&gt; 31,32已广泛用于消耗或淋巴细胞分离免疫领域12,31。这些技术的比较后，我们成立了目前的平移协议的TEC富集。在浓缩过程中的温和条件下会导致更少的细胞死亡和公正，提高TEC的恢复。 第3节中所描述的分离的胸腺细胞悬浮液可直接应用到流式细胞仪分析鉴定TEC的子集和树突状细胞和表征。第4节介绍了一个简单而有效的方法来确定使用多参数流式细胞仪TEC的子集。进行实验，寻求获得纯化的cTECs或mTECs，泰格丰富和细胞分选程序，可以在第5和第6被发现。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

胸腺是T细胞发展的重要器官。胸腺基质细胞，胸腺上皮细胞器（TEC）是在T细胞发育的多个阶段尤为关键：T细胞的承诺，正选择和负选择。然而，肾小管上皮在胸腺的功能尚未完全了解。在这篇文章中，我们提供了一种方法来隔离新鲜的小鼠胸腺采用机械破碎和酶消化相结合的TEC的子集。该方法允许胸腺基质细胞和胸腺细胞被有效地从细胞 - 细胞和细胞 - 细胞外基质的连接释放以形成单细胞悬浮液。使用分离的细胞，多参数流式细胞术可以应用于识别的TECs和树突状细胞和表征。由于TECs的是一种罕见的细胞群体在胸腺中，我们还描述了以丰富和消耗的胸腺细胞在胸腺中最丰富的细胞类型纯化的TECs的有效途径。 Follo翼的富集，细胞分选时可以减少这样的细胞生存力的丧失可以的TECs的纯化过程中被最小化。纯化细胞是适用于各种下游分析如实时PCR，Western blot和基因表达谱。该协议将促进TEC的功能的研究和以及体外 T细胞重建的发展。

使用该协议，胸腺器官是从成年小鼠（第2项）和胸腺细胞悬浮液中除去的制备如在第3节中概述将得到的细胞悬浮液组成的胸腺细胞，造血的基质细胞和非造血的基质细胞。 CD45是造血泛标记在两个胸腺细胞和造血的基质细胞，如巨噬细胞和树突状细胞中表达。相比之下，肾小管上皮是造血起源不是不表达CD45 15。细胞染色和流式细胞仪分析，进行与在第4节中概述。 有...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

在这里，我们描述了用于分离，鉴定和小鼠胸腺上皮细胞（TECs的）的纯化的有效方法。该协议可以用于胸腺功能的正常T细胞发育，胸腺机能障碍，和T细胞重建研究。

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-identification-purification-murine-thymic-epithelial

Research

JoVE Journal

Immunology and Infection

31.7K 次观看.  University of Minnesota. 在这里，我们描述了用于分离，鉴定和小鼠胸腺上皮细胞（TECs的）的纯化的有效方法。该协议可以用于胸腺功能的正常T细胞发育，胸腺机能障碍，和T细胞重建研究。 

视频: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

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 ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.