Bu araştırma AW, AHH genç araştırmacılar (GIF Young) için Alman İsrail Vakfı ve Dr ben İsrail Bilim AHH için Vakfı (No 1418/11) ve (No 1933/12) hibe tarafından desteklenmiştir . AHH ve AW için Diş Hekimliği İbrani Üniversitesi-Hadassah Okulu&#39;nda Cabakoff Araştırma Vakfı Fonu.

Önceden hazırlayın: <ul><li> PBS +% 2 FCS </li><li> Kollajenaz tip II ve DNAz Tip I, 1 mg / ml (örnek başına 1 mi) içinde 2 mg / ml &#39;si ile PBS +% 2 FCS </li><li> Steril cerrahi aletler </li><li> 0.5 M EDTA </li></ul> 1. Üst Dişeti Eksizyon Tekniği <ol><li> Onaylı IACUC rehberlik kullanarak fareler Euthanize. </li><li> Yanaklar ve keskin / künt düz makas ile mandibula ramus dahil olmak üzere ağız boşluğunun her iki kesin. </li><li> Çene aşağı çekin. </li><li> Standart makas ile üçte azı dişleri arkasında hayali bir çizgi 1 mm yumuşak ve sert doku kesin. Makas damak kemiği (Şekil 2A, mavi çizgiler) düzlemine dik yönlendirin. </li><li> Yumuşak ve sert doku kesici (Şekil 2A, mavi çizgiler) arkasında 2 mm insizyon. </li><li> Ağız ön kısmını aşağı çekin. Burun boşluğu bu aşamadan sonra gözlemlenmiştir. </li><li> Makas yerleştirindamak paralel, burun boşluğu içine bir bıçak koymak, diğer bıçak antre de insizyon gerekir. Posterior insizyon (Şekil 2A, yeşil hat) kadar kesin. Maksilla her iki tarafında bu adımı yineleyin. Sonunda maksilla kafatasının geri kalanı (Şekil 2B) ayrılır. </li><li> , Maksilla çıkarın orta sütür (Şekil 2, siyah kesikli çizgi) olarak kesilmiş ve alveol kemiğinin ulaşana kadar her hemi-maksilla damak dokusu kırpın. </li><li> Adson forseps (dişler olmadan) ile ön sınırından dişeti dokuları (iki hemi-üst çene) Peel. </li><li> PBS +% 2 FCS ile bir tabak içinde çıkarıldı dişeti yerleştirin. </li></ol> 2. Dişeti İşleme <ol><li> 1 ml PBS ile bir doku kültürü kabı (35 x 10 mm) eksize dişeti koyun +% 2 FCS, kollajenaz tip II ve DNAz Tip I&#39;in 1 mg / ml, 2 mg / ml &#39; </li><li> N ° 15 steril cerrahi bıçak ile de doku kıyma. </li><li> Trplakadan 15 ml konik bir tüp için doku ansfer. </li><li> 1 ml PBS +% 2 FCS ve aynı test tüpüne transfer ile plaka üzerinde kalan doku / hücre yıkayın. </li><li> 37 az 20 dakika bir çalkalayıcı inkübatör inkübe ° C, 200 rpm (öneri: ön ısıtma inkübatör). </li><li> 37 başka bir 10 dakika ° C, 200 rpm&#39;de 20 EDTA, 0.5 M ul ve inkübe ekleyin. </li><li> 12 PBS +% 2 FCS ile ml ve 4 santrifüj kadar test tüpüne doldurun ° C, 8 dakika boyunca 400 x g (öneri: ön soğuk santrifüj). </li><li> 2 ml PBS +% 2 FCS ile süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> 70 mikron hücre-süzgeç ile örnek filtre ve akışı kaydedin. </li><li> 4 ° C, 5 dakika boyunca 320 x g&#39;de santrifüjleyin. </li><li> 300 ul PBS +% 2 FCS ile süpernatant ve tekrar süspansiyon hücreleri çıkarın. </li><li> Hücreleri (yaklaşık 5-10 x 10 5 hücreleri, tek maksilla beklenen) sayın. </li></ol> 3. Hücre dışı ve Hücre içi AntikorSitometrisi Analiz Boyama Bu ışıklar kapalı, bir başlık içinde çalışmak tavsiye edilir. Bu, sürekli olarak soğuk bir ortamda hücreler tutmak, hem de gerekli olan tüm malzemeleri için çok önemlidir. <ol><li> 100 ul toplam hacim içinde 1 x 10 6 hücre başına her seçilmiş bir antikorun 0.2-0.5 ug eklenmesi ile hücre-dışı moleküllere karşı hücre Leke. </li><li> Vortex kısaca. </li><li> 4 ° C de karanlıkta 15 dakika süreyle inkübe edin </li><li> 2 ml PBS +% 2 FCS ile hücreleri yıkayın. </li><li> 4 ° C, 5 dakika boyunca 320 x g&#39;de santrifüjleyin. </li><li> Ekleyerek süpernatant ve tekrar süspansiyon hücreleri aspire, bilge bırak, 500 ul soğuk BD Cytoperm / Cytofix vorteks ise. </li><li> 4 ° C&#39;de 40 dakika karanlıkta C inkübe edin. </li><li> Vortex kısaca. </li><li> 1 ml soğuk BD Perm hücreler iki kez yıkayın / 4, 7 dakika boyunca 800 xg&#39;de Tampon ve sıkma yıkayın ° C </li><li> 300 ul dışında süpernatant aspire. </li><li> Tarafından hücre içinde hücre Leke100 ul toplam hacim içinde 1 x 10 6 hücre başına her seçilmiş bir antikorun 1 ug eklenmesi. </li><li> Vortex kısaca. </li><li> 4 ° C de karanlıkta 30 dakika boyunca inkübe </li><li> 1 ml soğuk BD Perm hücreler iki kez yıkayın / 4, 7 dakika boyunca 800 xg&#39;de Tampon ve sıkma yıkayın ° C </li><li> 300 ul soğuk BD Perm / Yıkama Tamponu ekleyerek süpernatant ve tekrar süspansiyon hücreleri aspire. </li><li> Vortex kısaca. </li><li> FACS tüpler için filtreleme süre süspansiyonu aktarın. </li><li> Analizi kadar karanlık ve soğuk bir ortamda tüpler tutun. </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>

Yazarlar ifşa hiçbir şey yok.

Tek bir fareden elde edilen üst çeneye gingival dokular, daha önce tarif edildiği gibi 1 hücre dışı ve hücre içi molekülleri ifade etmek için T ve B lenfositlerinin alt gruplar, hem de bunların özelliği analiz etmek için yeterlidir. Bununla birlikte, eğer nadir hücre popülasyonlarının (örneğin DC için) ilgilendiren, 2-3 fareler havuzu dokulara önerilir. Unutmayın ki, tercih, bu mümkündür sonra damak ve diş eti dokuları ve tüketim diş eti (adım 1.8-1.9 bir değişiklik) her ik...

Gingiva diş serviks kısmı çevreleyen yumuşak doku ve alveol (Şekil 1) kapsar. Dişeti daha mukozal epitel ve bağ dokusu (aynı zamanda submukozaya ya da lamina propria olarak da bilinir) ayrılabilir çiğneme mukoza türüdür. Dişeti ve komşu dişlerin anatomik yapısı bakteriler sürekli yerel bağışıklık sistemi meydan plak (biyofilm) ikamet ve geliştirmek için olanak sağlar. Periodontal hastalıkların 2 olarak adlandırılan bir durum - Sonuç olarak, enflamatuar yanıt, bazı durumlarda zararlı olur dişeti, gelişir. Temel olarak, plak indüklenen Periodontal patolojilerin gingivitis ve periodontitis ayrılabilir. Dişeti iltihabı dişeti ile sınırlı yerel inflamatuar yanıt bir geri durumu temsil eder. Periodontitis, diğer taraftan, geri dönüşü olmayan bir yıkıcı bir süreç olduğu bağlanma cihaz (alveol kemiği, periodontalbağ, sement ve diş eti) 3 yok edilir. Dişeti periodontal hastalıklar 4 sırasında olduğu gibi efektör ve endüktif her iki site hizmet etmek için önerilmiştir. İnsan çalışmalar dental plak yanıt olarak, immün efektör hücreleri ve molekülleri dinamik sızmış veya kalkış dişeti 5-7 olduğunu ileri sürmüşlerdir. Bu aktivite, periodontal yıkım 8,9 önemli bir rol oynadığı gösterilmiştir. Insan dokuları ile çalışan, bu patolojik sürecin ilgili değerli bilgiler sağlanan çalışmalar tarafından oluşturulan veri büyük, etik, teknik ve deneysel sınırlamalar sahip ise. Transgenik fareler istihdam yoluyla ve in vivo müdahaleler 10 neden-sonuç deneylerine izin deneysel modellerin geliştirilmesi. Sonuç olarak, periodontal hastalık ile ilgili mekanizmalar bilgimizi son yirmi yılda önemli ölçüde artmıştır. Buna rağmen, periodontal hastalıkların karmaşıklığı nedeniyle, oradadoku yıkımı kolaylaştıran yerel immün yanıt niteliği ile ilgili devam eden bir tartışma. Periodontal hastalıklar sırasında dişeti merkezi bağışıklık hücrelerinin işlevini anlayışımızda eksikliği vardır. Bu hastalık, gingivanın hedef dokuda oluşan patolojik inflamatuar etkinliklerini inceleme amaçlı olarak bu nedenle çok önemlidir.

<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

Biz tam olarak izole ve akım sitometri ve moleküler çalışmalar için fare dişeti doku işlemek için bir teknik geliştirdik. Dişeti bu periodontal hastalıklara neden olabilir sözlü biyofilm karşı konağın immün yanıtı yer olduğu için bağışıklık mekanizmaları incelemek için eşsiz ve önemli bir dokudur. Ayrıca, alveoler kemik dokusu için dişetinin yakın da enflamatuvar durumlarda kemik yeniden eğitim sağlayan. Bizim yöntemi daha önce 1 gösterdiği gibi Langerhans hücreleri ve T düzenleyici hücreleri gibi daha nadir hücre popülasyonlarının analizi sağlayan bağışıklık hücrelerinin büyük miktarda verir. Periodontal hastalıklar sırasında alveol kemik kaybı dahil yerel immün yanıtları incelemek için istihdam fareler nedeniyle çeşitli immünolojik ve deneysel araçların durumu avantajlıdır. Bununla birlikte, kendi küçük boyutu ve fare dişeti için nispeten uygunsuz erişim nedeniyle, birçok çalışma thi incelenmesi kaçınılmalıdırs kritik doku. Bu çalışmada açıklanan yöntemi umarım periodontal hastalıklar sırasında sözlü bağışıklık sistemi ve rolünü bizim understating artacak dişeti analizi, kolaylaştırabilir.

Dişeti hücreleri üzerinde akım sitometri analizi örnekleri sunulmaktadır. 2 farelerin toplanmış Dişeti hücreleri bir LSR II akış sitometresinde çalıştırın ve FlowJo yazılımı kullanılarak analiz edildi. Şekil 3A bir yan dağılım (SSC) karşı ileri dağılım (FSC) arsa içinde naif farelerden alınan dişeti hücrelerinin dağılımı göstermiştir. Tespit etmek Ayırıcı stratejisi, (i) lenfositler (ii) monositler / dendritik hücreler ve (iii) granülosit belirtilir. Karşıl...

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

Bu çalışma, bir tek hücre kültürü üretmek için fare ağız boşluğundan gingival dokular izole etmek ve işlemek için etkili bir yöntem açıklanır. Ortaya çıkan hücreler bir başka akış sitometri analizi ve klinik çalışmalar için kullanılabilir.

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

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

18.9K Görüntüleme.  Hebrew University - Hadassah Medical Center. Bu çalışma, bir tek hücre kültürü üretmek için fare ağız boşluğundan gingival dokular izole etmek ve işlemek için etkili bir yöntem açıklanır. Ortaya çıkan hücreler bir başka akış sitometri analizi ve klinik çalışmalar için kullanılabilir.

Video: 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.

İzolasyon, Karakterizasyonu ve Dişeti Bağışıklık Hücre Ağı Fonksiyonel Sınav

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

İmmünoloji ve Enfeksiyon

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.

Fare ve İnsan Dişlerinden Tek Hücrelerin Hızlı İzolasyonu

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

Gelişim Biyolojisi

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.

Fare Tükürük Bezi Kök Hücre İzolasyonu

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 ...

Biyoloji

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.

Oral Nötrofillerin Değerlendirilmesinde Mürin Periodontitin Sağlam Ligatür Kaynaklı Modeli

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 Kullanarak Birincil İnsan Dişeti Epitel Hücrelerinin İzolasyonu ve Kültürü

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

Biyomühendislik

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.

Murine glia'nın Multipl Merkezi Sinir Sistemi Bölgelerinden Diseksiyonu ve İzolasyonu

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

Nörobilim

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.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

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.

Yetişkin Murine lacrimal ve submandibular bezlerden Myoepitelyal hücrelerin izolasyonu

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.

<meta charset="utf8"></head><body>İntraperitoneal Bakteriyel Enfeksiyonun Murin Modelinde Splenik Mikroveziküllerin İzolasyonu

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.

İntraperitoneal Bakteriyel Enfeksiyonun Murin Modelinde Splenik Mikroveziküllerin İzolasyonu

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

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

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Y-27632 Kullanarak Birincil İnsan Dişeti Epitel Hücrelerinin İzolasyonu ve Kültürü

Murine glia'nın Multipl Merkezi Sinir Sistemi Bölgelerinden Diseksiyonu ve İzolasyonu

Murin Koroner Damar düz kas hücrelerinin izolasyonu

Yetişkin Murine lacrimal ve submandibular bezlerden Myoepitelyal hücrelerin izolasyonu

İntraperitoneal Bakteriyel Enfeksiyonun Murin Modelinde Splenik Mikroveziküllerin İzolasyonu

İntraperitoneal Bakteriyel Enfeksiyonun Murin Modelinde Splenik Mikroveziküllerin İzolasyonu