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 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

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

18.7K 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

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

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

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

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

İmmünoloji ve Enfeksiyon

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

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

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

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

Fare Periton Makrofagların izolasyonu Toll-benzeri reseptörler Uyarım üzerine Gen İfadesi Analizi Yapacak

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

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

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

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

İzolasyon ve Glioma-sızmakta periferik kan mononükleer hücrelerinin Akışı Sitometrik Analizi

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

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

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

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

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 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

İzolasyon ve murin lenfositlerin aktivasyonu

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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

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

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

İnsan Endoservikal Gamma Delta T Hücreleri İzolasyon ve Akım Sitometrisi Analizi

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

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

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

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

Lenfatik endotel hücreleri akış sitometrik çözümlenmesi için fare karaciğer sindirim

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

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

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

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

Yamultma akış kaynaklı geçiş antikorundan marjinal bölge B hücrelerinin in Vitro analizine

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