Name: isolation, processing and analysis of murine gingival cells
Uploaded: 2013-07-02T14:00:00.000+00:00
Duration: 9 min 47 s
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><tbody><tr><td>Collagenase Type II</td><td>Worthington Biochemical Corp.</td><td>CLS-2</td><td></td></tr><tr><td>DNAse I</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>EDTA</td><td>SIGMA</td><td>E6758-100G</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>Biological Industries</td><td>04-121-1</td><td>Heat Inactivated</td></tr><tr><td>Vacuum-Driven Filter</td><td>Jet Biofil</td><td>FPE-204-500</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>SPL Lifesciences</td><td>93070</td><td></td></tr><tr><td>Tissue Culture Dish 35&amp;#215;10 mm</td><td>NUNC</td><td>153066</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>Anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD45.2 antibody</td><td>Biolegend</td><td>109819</td><td>Clone 104</td></tr><tr><td>Anti-mouse CD4 antibody</td><td>Biolegend</td><td>100413</td><td>Clone GK1.5</td></tr><tr><td>Anti-mouse CD8a antibody</td><td>Biolegend</td><td>100733</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>Biolegend</td><td>100214</td><td>Clone 17A2</td></tr><tr><td>Anti-mouse CD326 (Ep-CAM) antibody</td><td>Biolegend</td><td>118219</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse CD207 (Langerin) antibody</td><td>Imgenex</td><td>DDX0362D</td><td>Clone 929F3.01</td></tr><tr><td>Anti-mouse I-A&lt;sup&gt;d&lt;/sup&gt; (MHC-II) antobody</td><td>Biolegend</td><td>115010</td><td>Clone 39-10-8</td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo Software v 7.6.5</td><td>Tree Star</td><td></td><td></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

18.8K 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

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 ...

科研

免疫与感染

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

从小鼠和人类牙齿中快速分离单个细胞

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

发育生物学

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

生物学

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

用于口腔中性粒细胞病评价的鼠期牙周炎的强结合诱导模型

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

生物工程

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

神经科学

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

用于研究厌氧菌-宿主细胞相互作用的细菌感染测定

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...