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

この研究は、イスラエル科学財団（番号1418から1411）からAHHと（番号1933から1912年）にAW、AHHに若い研究者のためのドイツのイスラエル財団（GIFヤング）、博士と私への補助金によって支えられてAHHとAWへの歯科医学のヘブライ大学 - ハダサスクールで。Cabakoff研究基金基金。

事前に準備します。 <ul><li> PBS + 2％FCS </li><li>コラゲナーゼタイプIIおよびDNアーゼタイプIの1 mg / mlの（サンプル当たり1ミリリットル）の2 mg / mlのとPBS + 2％FCS </li><li>滅菌手術器具</li><li> 0.5 M EDTA溶液</li></ul> 1。アッパー歯肉切除テクニック<ol><li>承認された動物実験委員会のガイダンスを使用して、マウスを安楽死させる。 </li><li>頬と鈍い/鋭いストレートハサミで下顎枝含む口腔の両側をカットします。 </li><li>下顎骨をプルダウン。 </li><li>標準ハサミで三分の臼歯の後ろに仮想線1ミリメートルでソフトとハードの組織を切り取る。はさみは口蓋骨の面（ 図2A、青い線）に垂直な演出。 </li><li>前歯の後ろにソフトとハードの組織2ミリメートル（ 図2A、青い線）を切開。 </li><li>口の前部をプルダウン。鼻腔は、この工程の後に観測される。 </li><li>はさみを置き口蓋と平行に、鼻腔内に1ブレードを入れて、他のブレードは前庭で切開する必要があります。後部切開（ 図2A、緑の線）までカットします。上顎の両側に、この手順を繰り返します。端部には上顎骨、頭蓋骨の残りの部分（ 図2B）から取り外される。 </li><li> 、上顎を削除ミドル縫合（ 図2、黒の破線）でそれをカットし、歯槽骨に達するまで、各半上顎の口蓋組織をトリミング。 </li><li>アドソン鉗子（歯なし）との前縁から歯肉組織（両方半上顎）をはがします。 </li><li> PBS + 2％FCSとプレートに切除歯肉を置きます。 </li></ol> 2。歯肉処理<ol><li> 1mlのPBSで組織培養皿（35×10㎜）で切除歯肉を入れ+ 2％FCS、コラゲナーゼタイプIIとタイプDNアーゼIの1 mg / mlの2 mg / mlの</li><li> N°15無菌手術用ブレードとよく組織をミンチ。 </li><li> TRプレートで15 mlコニカル試験管に組織をansfer。 </li><li> 1mlのPBS + 2％FCSと同じ試験管に移すと皿の上に残っている組織/細胞を洗浄。 </li><li> 37℃で20分間シェーカーインキュベーター内でインキュベート℃、200rpmで（推奨：予熱インキュベーター）。 </li><li> 37℃でさらに10分間℃、200rpmで、20 EDTA 0.5 Mの液とインキュベートを追加します。 </li><li> 12 PBS + 2％FCSとml及び4で遠心まで試験管を埋める°C、8分間400×gで（推奨：プレチル遠心分離機）。 </li><li> 2ミリリットルPBS + 2％FCSを上清と再懸濁した細胞を取り除きます。 </li><li> 70μmのセルストレーナーでサンプルをフィルタリングし、フロースルーを保存します。 </li><li> 4℃、5分間320×gで遠心。 </li><li> 300μlのPBS + 2％FCSを上清と再懸濁した細胞を取り除きます。 </li><li>細胞（約5〜10×10 5個の細胞が単一の上顎から期待されている）カウント。 </li></ol> 3。外および細胞内抗体フローサイトメトリー分析のための染色それは、ライトをオフにして、ボンネット内部の作業をすることをお勧めします。それは一定の寒い環境で細胞を維持するだけでなく、必要に応じてすべての材料が重要です。 <ol><li>総容量100μl中に1×10 6個の細胞あたりそれぞれ選択された抗体の0.2〜0.5μgのを加えることによって細胞外分子に対して細胞を染色。 </li><li>簡潔に渦。 </li><li> 4℃で暗所で15分間チューブをインキュベート</li><li> 2ミリリットルPBS + 2％FCSで細胞を洗浄します。 </li><li> 4℃、5分間320×gで遠心。 </li><li>ボルテックスしながら追加して上清と再懸濁した細胞を吸引、賢い、500μlの冷たいBD Cytoperm / Cytofixドロップします。 </li><li> 4℃で40分間チューブをインキュベートし、暗所でC。 </li><li>簡潔に渦。 </li><li> 1ミリリットル冷たいBDパーマで二回細胞を洗浄/ 4で7分間、800×gでバッファとスピンを洗う℃に</li><li> 300μlのを除いて、上清を吸引除去する。 </li><li>によって細胞内に細胞を染色100μlの総体積で1×10 6個の細胞あたりそれぞれ選択された抗体1μgのを追加。 </li><li>簡潔に渦。 </li><li> 4℃で暗所で30分間インキュベート</li><li> 1ミリリットル冷たいBDパーマで二回細胞を洗浄/ 4で7分間、800×gでバッファとスピンを洗う℃に</li><li> 300μlの冷たいBDパーマ/洗浄バッファを追加することで、上清と再懸濁した細胞を吸引。 </li><li>簡潔に渦。 </li><li> FACSチューブにフィルタリングしながらサスペンションを移す。 </li><li>分析まで暗くて寒い環境の中で管を保つ。 </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>

著者らは、開示することは何もありません。

単一のマウスから得られた上顎歯肉組織は、我々は以前に説明したように1外および細胞内の分子を発現するTリンパ球およびBリンパ球の亜集団、ならびにそれらの能力を分析するために十分である。希少細胞集団が興味（例えばDCの）である場合にもかかわらず、それは2-3マウスからプール組織に推奨されます。望ましい場合に注目すべきは、それが歯肉（ステップ1.8から1.9の変更）...

歯肉は、歯頸部の周囲の軟部組織である歯槽プロセス（ 図1）をカバーしています。歯肉はさらに、粘膜上皮と結合組織（また、粘膜または粘膜固有層とも呼ばれる）に分けることができる咀嚼粘膜の一種である。歯肉と隣接する歯の解剖学的構造は、細菌が常にローカル免疫系に挑戦プラーク（バイオフィルム）が存在し、開発することができます。結果として、炎症反応は、特定の状況では破壊的になる歯肉に発症-条件は、歯周病2と呼ば。基本的には、歯垢誘発性歯周病態は、歯肉炎と歯周炎に分けることができます。歯肉炎は歯肉に限定されている地元の炎症反応の可逆的な状態を表しています。歯周炎、その一方で、不可逆的な破壊的なプロセスである取付装置（歯槽骨、歯周靭帯、セメント質と歯肉）は3を破壊している。 歯肉は歯周病4時などのエフェクターや誘導サイトの両方を提供することを提案した。ヒトでの研究は、歯垢に対応して、免疫エフェクター細胞および分子が動的に浸潤または出発歯肉5-7を示唆している。この活性は、歯周破壊8,9において主要な役割を果たすことが示された。ヒト組織と協力し、この病理学的過程に関する貴重な情報を提供し、それらの研究によって生成されたデータは、主要な、倫理的、技術的、実験的な限界を持っているのに対し。トランスジェニックマウスを用いてビアおよびin vivoでの介入10因果実験法を許可した実験モデルの開発。その結果、歯周病に関与するメカニズムに関する我々の知識は、最後の二十年の間に大幅に増加した。そうであっても、歯周病の複雑さに起因がある組織破壊を促進する局所免疫応答の性質に関する進行中の議論。歯周病の間の歯肉の中心的な免疫細胞の機能に関する我々の理解に不足があります。それは病気、歯肉の標的組織で発生する病理学的炎症事象を研究することが不可欠です。

<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

我々は正確にフローサイトメトリーや分子の研究のためにネズミ歯肉組織を分離して処理するための技術を開発した。歯肉は、それが歯周病の原因となることがあり、口腔バイオフィルムに対する宿主免疫応答に関与しているため、免疫機構を研究するためのユニークで重要な組織である。さらに、歯槽骨組織への歯肉のすぐ近くには、炎症性の条件下で骨リモデリングを勉強できます。本手法は、我々は以前に1を示したようなランゲルハンス細胞およびT調節細胞でさえ希少細胞集団の分析を可能にする免疫細胞を大量に得られる。歯周病の間に歯槽骨の損失に関わる局所免疫応答を研究するためにマウスを採用するため、様々な免疫学的および実験的なツールの利用可能性に有利である。それにもかかわらず、彼らの小さなサイズとネズミ歯肉に比較的不便なアクセスによる、多くの研究は、THIの検査を避けたの重要な組織。この作品に記載された方法がうまくいけば、歯周病の間に私達の経口免疫システムに過小とその役割が大きくなり歯肉分析を容易にすることができます。

歯肉細胞に対するフローサイトメトリー分析の例が示されている。 2匹のマウスからのプールされた歯肉細胞をLSR IIフローサイトメーターで実行され、FlowJoソフトウェアを用いて分析した。 図3A側方散乱（SSC）対前方散乱（FSC）プロットにおけるナイーブマウスから歯肉細胞の分布を示した。識別するためのゲーティング戦略は、（i）リンパ球（ii）の単球/樹状細胞および（iii?...

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

この研究は、単一細胞培養を生成するために、マウス口腔から歯肉組織を分離して処理するための効率的な手法が記載されている。得られた細胞をさらにフローサイトメトリー分析および分子生物学的研究のために使用することができる。

マウス歯肉細胞の単離、処理と解析

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 ビュー.  Hebrew University - Hadassah Medical Center. この研究は、単一細胞培養を生成するために、マウス口腔から歯肉組織を分離して処理するための効率的な手法が記載されている。得られた細胞をさらにフローサイトメトリー分析および分子生物学的研究のために使用することができる。

ビデオ: 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.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

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

免疫・感染学

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.

マウスとヒトの歯からの単一細胞の迅速な分離

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

発生生物学

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.

マウス唾液腺幹細胞の単離

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.

口腔好中球評価のためのマウス歯肉炎の堅牢な合字誘発モデル

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.

Y-27632を用いた初原ヒト歯肉上皮細胞の単離と培養

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

生物工学

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.

複数の中枢神経系領域からのマウスグリアの解剖と分離

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

神経科学

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.

マウス冠動脈血管平滑筋細胞の単離

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.

成人ネズミ涙腺および顎下腺からの筋上皮細胞の単離

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.

嫌気性細菌-宿主細胞相互作用を研究するための細菌感染アッセイ

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