该研究得到了补助，以色列科学基金会（第1418/11号）AHH（第1933/12号）AW，年轻的研究者（GIF年轻）的德国以色列基金会AHH，我博士希伯来大学哈达萨学校的牙科医学AHH​​和AW Cabakoff研究基金。

提前准备： <ul><li> PBS + 2％FCS </li><li> PBS + 2％FCS与胶原酶II型和1毫克/毫升的DNA酶I型（1毫升，每样2毫克/​​毫升） </li><li>消毒手术器械</li><li> 0.5 M EDTA溶液</li></ul> 1。上牙龈切除技术<ol><li>安乐死小鼠使用批准IACUC指导。 </li><li>切口腔两侧脸颊和下颌支与锐/钝直剪刀。 </li><li>拉下颌骨。 </li><li>切割软硬组织在一个假想的线1毫米标准剪刀三分之二磨牙后面。直接剪刀垂直于腭骨的平面内（ 图2A中 ，蓝色的线）。 </li><li>切开软，硬组织2毫米背后的门牙（ 图2A，蓝色的线）。 </li><li>拉下嘴的前部。此步骤之后，可观察的鼻腔。 </li><li>将剪刀与上腭平行，把一个刀片进入鼻腔，其他刀片切开前庭。切，直到后切口（ 图2A，绿线）。上颌骨两侧，重复此步骤。在结束上颌骨脱离颅骨的其余部分（ 图2B）。 </li><li>取下颌骨，切在中间缝合（ 图2，黑色虚线），直到达到牙槽骨修剪每个半侧颌骨的腭组织。 </li><li>剥离牙龈组织（半侧颌骨）从他们的前缘阿德森钳（无齿）。 </li><li>将切除牙龈用PBS + 2％FCS一盘。 </li></ol> 2。牙龈处理<ol><li>将切下的牙龈组织培养皿（35×10毫米），用1ml PBS + 2％FCS，2毫克/毫升胶原酶II型和1毫克/毫升DNA酶I型</li><li>百果组织的N°15无菌手术刀片。 </li><li>风帆ansfer组织从板到15毫升锥形试管中。 </li><li>盘上剩余的组织/细胞洗净，用1ml PBS +2％FCS中，转移到相同的试管。 </li><li>在摇床培养箱孵育20分钟，37℃，200转（建议：预热孵化）。 </li><li>加入20微升0.5 M EDTA和孵化，再过10分钟，在37°C，200转。 </li><li>在试管至12毫升，用PBS + 2％FCS和离心机在4°C，400 XG 8分钟（建议：预冷离心机）。 </li><li>取出上清，重悬细胞2毫升PBS + 2％FCS。 </li><li> 70微米的细胞过滤器和过滤器样品，节省流量通过。 </li><li>离心机在4℃，320×g离心5分钟。 </li><li>上清，重悬细胞取出300微升PBS + 2％FCS。 </li><li>计数细胞（约5×10 5细胞从一个单一的上颌骨）预期。 </li></ol> 3。细胞外和细胞内抗体染色流式细胞仪分析建议里面工作引擎盖，灯熄灭。这是至关重要的，以保持细胞在恒冷的环境中，以及所需的所有材料。 <ol><li>每1×10 6个细胞的总体积为100μl，加入0.2-0.5微克每个选定的抗体染色的细胞对细胞外分子。 </li><li>涡简要介绍。 </li><li>孵育15分钟，在黑暗中在4℃下在管</li><li>洗涤细胞2毫升PBS + 2％FCS。 </li><li>离心机在4℃，320×g离心5分钟。 </li><li>吸上清，重悬细胞，加入，逐滴，500微升冷BD Cytoperm / Cytofix的而震荡。 </li><li>孵育40分钟，在4℃下在黑暗中管。 </li><li>涡简要介绍。 </li><li>洗涤细胞两次与1毫升冷BD烫发/洗涤缓冲和自旋在800 XG 7分钟在4°C。 </li><li>吸上清液300μl的除外。 </li><li>细胞染色细胞每个选定的抗体加入1微克每1×10 6个细胞在总体积为100μl。 </li><li>涡简要介绍。 </li><li>在4℃下孵育30分钟，在黑暗</li><li>洗涤细胞两次与1毫升冷BD烫发/洗涤缓冲和自旋在800 XG 7分钟在4°C。 </li><li>吸上清，重悬细胞，加入300μL冷BD的彼尔姆/洗涤缓冲液。 </li><li>涡简要介绍。 </li><li>暂停转移，同时过滤FACS管。 </li><li>保持管在黑暗和寒冷的环境中，直到分析。 </li></ol>

<ol>
	<li>Arizon, M., 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=Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss.">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li><li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, R., 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=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., 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=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

作者什么都没有透露。

颌骨牙龈组织从一个单一的鼠标得到足够的用于分析的T和B淋巴细胞的子种群，以及他们的能力，表达细胞外和细胞内的分子，如我们之前描述1。然而，如果还罕见的细胞种群（例如区议会），它是建议到池组织从2-3只小鼠。值得注意的是，如果优选的话，它有可能剥离腭和牙龈组织，然后切除牙龈（修改步骤1.8-1.9）。还应该提到，下颌牙龈，以及可以收集的是，它的隔离是更复杂的，?...

牙龈是宫颈部的牙齿周围的软组织和覆盖肺泡的过程（ 图1）。齿龈是一种咀嚼粘膜，粘膜上皮和结缔组织（也被称为粘膜下层或固有层），并且可以进一步分离成不同的。牙龈和牙齿相邻的解剖结构，使细菌居住和发展斑块（生物膜）局部免疫系统，不断挑战。其结果是，炎症反应的发展的牙龈，这在某些情况下变得有破坏性的-这种情况称为牙周疾病2。基本上可分为牙龈炎和牙周炎，牙斑引起的牙周病理学。牙龈炎局限于牙龈的局部炎症反应是一个可逆的条件。牙周炎，在另一方面，是一种不可逆的破坏过程，其中所述附接装置（牙槽骨，牙周韧带，牙骨质和牙龈）被摧毁3。 牙龈提出服务效应和感性的网站，在牙周疾病4。人类的研究表明，在应对牙菌斑，免疫效应细胞和分子动态渗透或出发的牙龈5-7。这项活动被证明发挥了重要作用，在牙周破坏8,9。鉴于这些研究提供了有价值的信息，关于这个病理过程，与人体组织所产生的数据具有重大的伦理，技术和实验的局限性。开发实验模型允许因果experimentations通过采用转基因小鼠体内干预10。因此，我们的知识在牙周疾病所涉及的机制大大增加，在过去的二十年。即便如此，由于牙周疾病的复杂性，有正在进行的辩论，关于促进组织破坏局部免疫反应的性质。还有一个在我们的理解缺乏牙龈牙周疾病的中枢免疫细胞的功能。因此，必须研究病理在靶组织的疾病，牙龈炎症发生的事件。

<table border="1">
 <tbody>
 <tr>
 <td>Comments (optional)</td>
 <td>Catalogue number</td>
 <td>Company</td>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td></td>
 <td>CLS-2</td>
 <td>Worthington Biochemical Corp.</td>
 <td>Collagenase Type II</td>
 </tr>
 <tr>
 <td></td>
 <td>DN25-1G</td>
 <td>SIGMA</td>
 <td>DNAse I</td>
 </tr>
 <tr>
 <td></td>
 <td>E6758-100G</td>
 <td>SIGMA</td>
 <td>EDTA</td>
 </tr>
 <tr>
 <td></td>
 <td>D8537</td>
 <td>SIGMA</td>
 <td>Dulbecco's PBS</td>
 </tr>
 <tr>
 <td>Heat Inactivated</td>
 <td>04-121-1</td>
 <td>Biological Industries</td>
 <td>Fetal Bovine Serum </td>
 </tr>
 <tr>
 <td></td>
 <td>FPE-204-500</td>
 <td>Jet Biofil</td>
 <td>Vacuum-Driven Filter</td>
 </tr>
 <tr>
 <td></td>
 <td>352052</td>
 <td>BD Falcon</td>
 <td>5 ml Polystyrene Round-Bottom Tube</td>
 </tr>
 <tr>
 <td></td>
 <td>93070</td>
 <td>SPL Lifesciences</td>
 <td>Cell Strainer 70 &mu;m</td>
 </tr>
 <tr>
 <td></td>
 <td>153066</td>
 <td>NUNC</td>
 <td>Tissue Culture Dish 35&times;10 mm</td>
 </tr>
 <tr>
 <td></td>
 <td>554714</td>
 <td>BD</td>
 <td>BD Cytofix/Cytoperm</td>
 </tr>
 <tr>
 <td>Clone N418</td>
 <td>117305</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD11c antibody</td>
 </tr>
 <tr>
 <td>Clone 104</td>
 <td>109819</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD45.2 antibody</td>
 </tr>
 <tr>
 <td>Clone GK1.5</td>
 <td>100413</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD4 antibody</td>
 </tr>
 <tr>
 <td>Clone 53-6.7</td>
 <td>100733</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD8a antibody</td>
 </tr>
 <tr>
 <td>Clone 17A2</td>
 <td>100214</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD3 antibody</td>
 </tr>
 <tr>
 <td>Clone G8.8</td>
 <td>118219</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD326 (Ep-CAM) antibody</td>
 </tr>
 <tr>
 <td>Clone 929F3.01</td>
 <td>DDX0362D</td>
 <td>Imgenex</td>
 <td>Anti-mouse CD207 (Langerin) antibody</td>
 </tr>
 <tr>
 <td>Clone 39-10-8</td>
 <td>115010</td>
 <td>Biolegend</td>
 <td>Anti-mouse I-Ad (MHC-II) antobody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>BD Biosciences</td>
 <td>LSR II Flow Cytometer</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>Tree Star</td>
 <td>FlowJo Software v 7.6.5</td>
 </tr>
 </tbody>
</table>

isolation, processing and analysis of murine gingival cells

我们已经开发出一种技术，能够精确地隔离和处理流式细胞术及分子生物学研究的小鼠牙龈组织。牙龈是一个独特而重要的组织来研究，因为它是免疫机制参与宿主的免疫反应，可能会导致牙周疾病对口腔生物膜。此外，靠近牙龈牙槽骨组织，也能学习骨重塑炎症条件下。我们的方法会产生大量的免疫细胞，允许甚至罕见的细胞群，如朗格汉斯细胞和调节性T细胞的分析，为大家展示了以前1。用人小鼠研究参与齿槽骨丧失在牙周疾病的局部免疫反应是有利的，因为各种免疫学和实验工具的可用性。然而，由于其体积小，小鼠牙龈相对交通不便，许多研究避免氏检查关键组织。在这项工作中所描述的方法可促进牙龈的分析，希望将增加我们在牙周疾病的口服免疫系统，它的作用低估。

作为对牙龈细胞的流式细胞仪分析。汇集从2小鼠牙龈细胞运行在一个LSR II流式细胞仪，使用FlowJo软件分析表明牙龈细胞的分布图3A从一个侧面散射（SSC）与前向散射（FSC）情节天真的小鼠。选通策略，以确定（一）淋巴细胞（ⅱ）单核细胞/树突状细胞，粒细胞及（iii）表示。比较而言，我们还提出了流式细胞仪示出了牙龈细胞纯化P.牙龈卟啉单胞菌感染小鼠灌胃后21天在一个设...

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

本研究中描述了一种有效的技术，用于隔离和处理，以产生一个单一的细胞培养的小鼠口腔牙龈组织。得到的细胞可进一步用于流式细胞分析和分子水平的研究。

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-processing-and-analysis-of-murine-gingival-cells

Research

JoVE Journal

Immunology and Infection

18.7K 次观看.  Hebrew University - Hadassah Medical Center. 本研究中描述了一种有效的技术，用于隔离和处理，以产生一个单一的细胞培养的小鼠口腔牙龈组织。得到的细胞可进一步用于流式细胞分析和分子水平的研究。

视频: Isolation, Processing and Analysis of Murine Gingival Cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

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 of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...