Name: isolation, processing and analysis of murine gingival cells
Uploaded: 2013-07-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

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...

<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>

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 ...

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

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

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

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 Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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

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

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

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

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

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 ...