Yazarlar E1E4 antikor tür hediye için Sally Roberts (University of Birmingham, Birmingham UK) teşekkür etmek istiyorum. Bu çalışma, Ulusal Kanser Enstitüsü (4R00CA137160-03) bir hibe ile desteklenmiştir.

1. Organotipik Raft Kültürler Hazırlık <ol><li> Metal sal ızgaraları kadar ilk farklılaşma sürecine müdahale olabilecek artıkları temizlemek için kromik sülfürik asit ile tedavi edilebilir. Bir saat için sülfürik asit içeren bir cam beher içinde bırakın metal ızgaralar, daha sonra sürekli musluk suyu ile bir gece durulanmalıdır. Durulama gece sonra, radye ızgaraları çift distile su içinde 3-5 saat durulanmalıdır. </li><li> Sal destek sağlamak için, birbirinden eşit mesafede 0.5 cm radye üç tarafı bükün. Metal ızgaraları sonra otoklava edilmelidir. </li><li> 2,2 g NaHCO3 ve 4.8 g Hepes ekleyerek 10X sulandırma tamponu hazırlayın. 0.05 M NaOH 100 ml eritin. Filtre sterilize ve -20 ° C&#39;de 5 ml hacimde depolamak </li><li> Sodyum bikarbonat olmadan 10X DMEM hazırlayın. Filtre sterilize ve -20 ° C&#39;de 5 ml hacimde depolamak </li><li> Fare epidermal büyüme faktörü hazırlayın: EGF 100 mg ve B 10 mg eritilirSA 10 ml her GKD 2 0 deiyonize. EGF ve BSA birleştirin ve 100 ml toplam hacim deiyonize GKD 2 0 80 ml ​​ekleyin. Filtre sterilize, tablet (5 ml) ve -20 ° C&#39;de saklayın E, orta her litre için 1 mg / ml EGF (nihai konsantrasyon 5 ng / ml), 5 ml ilave edilir. </li></ol> 2. Kollajen Jeller hazırlanması <ol><li> Bir kollajen jel her salı için gereklidir. Kollajen katılaşabilme önlemek için buz üzerinde tutulmalıdır. Sıçan soğuk-kuyruk kolajeni tip I (nihai konsantrasyon 3-4 mg / ml), 0.3 ml 10X rekonstitüsyon tamponu, 0.3 ml DMEM 10X AND1-2 X 10 6 fare 2.4 ml: her kollajen jel oluşan kollajen karışımı 3 ml gerektirir besleyici hücreler olarak 3T3 J2 fibroblastlar. Fare kuyruk kolajeni ile yüksek konsantrasyonlu çözümler için, doğru çalışma konsantrasyonu elde etmek için 0.02 M asetik asit içinde tamamlanır. </li><li> Eğer kolajen miktarı, 10X reconstit için bir master miks hesaplamak daha sonra ihtiyacınız olacak kollajen jel (sallar) sayısını belirlemekution tampon, 10X DMEM ve J2 fibroblastlar gereklidir. Kollajen viskozite nedeniyle 3-4 ekstra jeller için her ana karışım yeterli ekleyin. </li><li> Kollajen jel hazırlamak için, J2 fibroblastlar trypsinize ve orta ile nötralize. 50 ml&#39;lik konik flakon tüm fibroblastlar ve yerde birleştirin. Yapılabilir sallar numarasını belirlemek için hücreleri hesaplama. Yine, 1-2x10 6 fibroblastlar sal başına böylece kollajen jel başına gereken ve uygulanır. Düşük hızda hücreleri aşağı Spin. </li><li> 10X rekonstitüsyon tamponu, 10X DMEM ve hesaplanan salları sayısı için kolajen uygun miktarlarda fibroblast pelet ve Pastör pipetiyle gelen ortamı aspire. Hızlı katılaşmayı gelen jel önlemek için kolajen son ekleyin. Kollajen eklendikten sonra, jel içine kabarcıklarının giriş önlemek için hızlı bir şekilde yavaşça karışımı, fakat. Jel Doğru pH göstergesi kırmızımsı portakal rengi, olmalıdır. Jel karışımı aşırı sarı ise, filtre ile sterilize 1N damla bir çift eklemeNaOH ve karışımı yavaşça doğru renk elde etmek. </li><li> Kollajen 3 ml ekleyin: fibroblast karışımı 6 kuyu kültür yemeğin kuyulara. Kollajen karışımı kabarcıklar oluşturarak önlemek için yavaş yavaş her iyi tarafı aşağı pipetlenir edilmelidir. Bir 37 ° C doku kültürü plakası inkübatörde yerleştirin ve 30 dakika boyunca katılaşmaya izin verir. </li><li> 30 dakika sonra, katılaştırılmış kollajen jel üst EGF E orta 3 ml ekleyin ve plaka inkübatöre geri döner. Jeller, en az bir gün boyunca 37 ° C&#39;de tutulur, ancak dört gün içinde kullanılmalıdır. Optimal sonuçlar inkübasyon iki gün sonra ortaya çıkar. </li></ol> 3. Farklılaşma için hazırlık Keratinositler <ol><li> Düşük geçit keratinositler yaklaşık% 70 konfluense büyüdü edilmelidir. Her salı 1-2x10 6 keratinositler gerektirir. Keratinositler Trypsinize ve EGF ile E orta ile nötralize. Yapılabilir sallar numarasını belirlemek için hücreleri hesaplama. Düşük SPE olarak hücreleri aşağı Spined. Hesaplanan her sal kültürü için EGF ile E ortamın 1 ml pelletini. </li><li> Eğerek 6 sıra plaka ile kollajen jel gelen orta aspire. Her jel EGF taze E orta 2 ml ekleyin. Her jel için, dikkatle de zaten mevcut E orta 2 ml süspanse keratinositlerin 1 ml ekleyin. Sonra yavaşça pipet keratinositlerin artı medyanın 2 ml ve sonbahar açılan akıllıca geri jel üzerine izin verin. Çalkalama veya plaka sallanan kollajen jel bir tarafında hücrelerinin düzensiz dağılımı ile sonuçlanabilir. 37 ° C inkübatörde plakası yerleştirin. </li><li> Hücreler günlük E orta yerine, konfluent için yetiştirilen edilmelidir. Hücreler konfluent olduğunda, orta bir değişiklikten sonra sarı bir gün için orta değişir. Bu genellikle 2-4 gün sürer. Orta günde dört tarafından değişmedi, bir sonraki adıma geçin. </li></ol> 4. Radye Kültürler yapma <ol><li> Steril bir metal sal ızgara yerleştirmek için steril forseps kullanma, taraf eğildi, Bir 10 cm çanak içine. Kollajen jelden orta aspire. İyi bir taraftan jel gevşetmek için, hareketli bir yukarı ve aşağı kullanılarak steril bir spatula ile jel çevresi etrafında gider. Kollajen jel kaldırmak için hafifçe eğin plaka ve bir spatula altına koyarak kaldırın. Izgara ve jel arasındaki kabarcıklar oluşturmadan metal ızgara üzerinde kollajen jel yatırın. İki jeller her ızgara üzerine yerleştirilebilir. </li><li> Bir hava-sıvı arayüz hale getirmek için, sal ızgara ortam dokunmadan böylece tabağın tabanına kadar yavaş EGF olmadan E orta ilave fakat kollajen jel değildir. Medya kılavuzunda deliklerden gelmemeli. Bu tip farklılaşmasını önlemek olacak şekilde, kılavuz altında kabarcıkları üretmek önlemek için yavaş yavaş ortam ekleyin. 37 ° C&#39;de inkübe sallar ve hava-sıvı arayüzü koruyarak her geçen gün orta değiştirin. 14 gün sonra hasat sallar. </li></ol> 5.. Temsilcisi Sonuçlar Zaman tO protokolü primer keratinositler ile düzgün bir şekilde gerçekleştirilir, bu işlem cilt katman değişik belirgindir iyi farklılaşmış epitel neden olacaktır. Bu Şekil 1B gösterildiği hematoksilen ve eozin (H &amp; E) sal kültürler, bölümleri boyanarak histolojik inceleme ile elde edilebilir. H &amp; E boyama sallar ve tabakalandırmak ve ayırt hücrelerinin yeteneği üzerinde virüs veya antiviral bileşiklerin etkisi morfolojisini incelemek de faydalıdır. Örneğin, HPV-ölümsüzleştirdi birincil insan keratinositleri (HFK-31) yapılan sallar özellikle kalın çoğalması oranında herhangi bir artış ve farklılaşma hücreleri aktif tutmak için HPV proteinlerin yeteneğini yansıtan, normal insan sünnet derisi keratinositleri (HFK) yapılan sallar karşılaştırıldığında hücre döngüsü içinde (Şekil 1B) 10. Epitel boyunca çekirdeklerin tutulması Bu sonuç, normal keratinositlerin bir arıza ile sonuçlanan farklılaşma üzerine hücre döngüsü çıkmak iseçekirdek zarı. Daha yakından incelemek için farklılaşma, immünhistokimya Sitokeratin yanı sıra involukrin ve filagrin gibi özel farklılaşma belirteçler, incelemek için yapılabilir. Farklı Sitokeratin farklılaşma 7 spesifik aşamaları ile ifade edilir. Şekil 2 de gösterildiği gibi, sitokeratin 10 (K10) taban katmanı olarak ifade değildir ve tek aşamalı olarak ayırt hücreleri içerir suprabasal tabakalar halinde bulunur. HPV pozitif sallar olarak, K10 ifadesi tekrar keratinosit farklılaşma HPV proteinlerinin etkisini yansıtan Normal keratinositler kıyasla gecikir. İmmünohistokimya da viral genlerin ifadesi profili incelemek için kullanılabilir. Şekil 3&#39;te gösterildiği gibi ifade epitelinin üst tabaka ile sınırlıdır geç bir proteindir HPV proteini E1 ^ E4, ifade inceleyerek bir temsilcisi olan boyama. Beklenildiği gibi, herhangi bir boyama, normal huma gelen sal kültürlerinde görülmektedirSünnet derisi keratinositler n. <img alt="Şekil 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> Şekil 1. Primer keratinositler ya epitel hücre hattından organotipik sal kültürler hazırlanması için yöntem A) içeriği. Stably HPV-31 (HFK-31) episomes veya normal insan sünnet derisi keratinositleri (HFK) den korumak, insan sünnet derisi keratinositlerinden oluşturulan organotipik sal kültür B) Hematoksilen ve eozin boyanma. Bireysel epiteliyal tabakalar yanı kollajen tıkaç olarak tespit edilir. <img alt="Şekil 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> Şekil 2. Sitokeratin 10 (K10) ifade epitelin suprabasal katmanları ile sınırlıdır. İmmünohistokimya K10 için bir antikor kullanılarak HFK-31 hücreleri, hem de normal bir HFKs elde organotipik sal kültürlerin kesitleri üzerinde yapıldı. Hücresel DNA DAPI ile zıt edildi. Görüntüler conf kullanılarak çekilenÖcal floresan mikroskobu. Oklar epitelin bazal tabaka göstermektedir. <img alt="Şekil 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> Şekil 3. HPV E1 ^ E4 proteini geç virüs ömrü çevrimi ile verimli bir faz olarak üretilir. İmmünohistokimya E1E4 karşı antikorlar kullanılarak HFK-31 hücreleri, hem de normal bir HFKs elde organotipik sal kültürlerin kesitleri üzerinde yapıldı. Hücresel DNA DAPI ile zıt edildi. Görüntüler konfokal floresan mikroskobu kullanarak ele geçirildi. Oklar epitelin bazal tabaka göstermektedir.

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. 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+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

Biz ifşa hiçbir şey yok.

Burada genel olarak epitelyal farklılaşma incelemek için kullanılabilecek bir yöntem tarif eder, fakat, aynı zamanda kolaylıkla viral patogenezine gibi potansiyel terapötik etkinlik incelemek için adapte edilebilir. Birçok virüs herpesvirüslerinin ile olduğu gibi, HPV durumunda olduğu gibi, ya da virüs ömrü çevrimi içinde bir noktada, enfeksiyon primer olarak ya epitel hücreleri hedef. Konak hücre etkileşimleri: organotipik sal kültürler büyüyen zaman alıcı, virüsü incelemek için son derec...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Sıçan Kuyruk Kollajen tip 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> NaHC0 3 olmadan DMEM </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> Paslanmaz çelik metal ızgaralar </td><td> Williams ve hırs A.Ş. </td><td> CR-03063-040-100-S </td></tr><tr><td> E orta 11 </td><td></td><td></td></tr><tr><td> Epidermal Büyüme Faktörü </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

Organotipik epitel sal kültürlerin gelişimi vitro sistemde sadakatle özetlediği epitel farklılaşmasında etkili olan araştırmacılar sağlamıştır. Bu sistem için bir çok kullanıma vardır. Örneğin, keratinositlerin üç boyutlu organotipik sal kültürleri yetiştirme yeteneğiyle, insan papilloma virüsü (HPV) 1 çalışmalarında önemli bir kilometre taşı olmuştur. HPV yaşam döngüsü sıkıca skuamöz epitel 2 farklılaşma bağlantılıdır. Olarak Organotipik epitel sal kültürler burada virüs üretimi 3,4,5 dahil olmak üzere, tüm papillomavirus yaşam döngüsü yeniden gösterdi. Ayrıca, bu sal kültürler HPV ile in vivo enfeksiyon üzerine görülenlere benzerdir displastik lezyonlar gösterirler. Böylece bu sistem, aynı zamanda epitel hücre kanserleri, yanı sıra genel olarak epitel hücre farklılaşması üzerinde ilaçların etkisini araştırmak üzere de kullanılabilir. Başlangıçta Asselineau ve Prunieras tarafından geliştirilen <s6&gt; yukarı ve Kopan ve ark. 7 tarafından değiştirilmiş, organotipik epitel sal kültürü sistemi üzerindeki kolajen hücrelerinin büyümesini içeren bir genel olarak, nispeten kolay kültürü modeli, haline geldi bir hava-sıvı arayüzü (Şekil 1A) muhafaza bağlanır. 10-14 gün boyunca, hücrelerin tabakalandırmak ve farklılığı, farklılaşma özgü Sitokeratin üreten bir tam kat epitel oluşturulması. Hasat sal yanı sıra standart moleküler ve biyokimyasal teknikler ile, histolojik olarak kontrol edilebilir. Bu yazıda, birincil insan keratinositlerinden sal kültürlerin üretimi için bir yöntem açıklanmaktadır. Aynı teknik kurulan epitel hücre çizgileri ile birlikte kullanılabilir, ve kolay bir şekilde, normal veya hastalıklı biyopsileri 8 den epiteliyal doku ile kullanım için uyarlanabilir. Birçok virüs hedef ya deri veya mukozal epitelin kendi replikatif yaşam döngüsünün bir parçası olarak. Son birkaç yılda, fizibilite organotipik sal kült kullanarakVirüs-konak hücre etkileşim okuyan bir yöntem olarak ures birkaç herpesvirüslerinin yanı sıra adenovirüsler, parvoviruses ve poxviruses 9 için gösterilmiştir. Organotipik sal kültürler böylece viral patogenez incelemek için uyarlanmış ve kalıcı hücre serilerinde tarıma olmayan bu virüs için antiviral ajanlarla test etmek için tek yöntem olabilir.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Bir<em&gt; In vitro</emTaklit etmek&gt; yöntemi<em&gt; In vivo</em&gt; Epitelyal farklılaşma tarif edilmektedir. Birçok virüs bulaşıcı yaşam döngüsünün bir parçası olarak epitel hücreleri hedef ve bu yöntem, virüsün incelenmesi için bir araç sağlar: konak etkileşimleri daha yakından oluştuğu benzediği<em&gt; In vivo</em&gt;. Bu tekniğin temel keratinositler, oluşturulan hücre çizgileri, aynı zamanda, normal veya hastalıklı biyopsi doku ile birlikte kullanılabilir.

İlköğretim İnsan Keratinositler gelen Organotipik Radye Kültürlerin Üretimi

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.0K Görüntüleme.  University of North Carolina-Chapel Hill. Bir In vitro yöntemi In vivo Epitelyal farklılaşma tarif edilmektedir. Birçok virüs bulaşıcı yaşam döngüsünün bir parçası olarak epitel hücreleri hedef ve bu yöntem, virüsün incelenmesi için bir araç sağlar: konak etkileşimleri daha yakından oluştuğu benzediği In vivo. Bu tekniğin temel keratinositler, oluşturulan hücre çizgileri, aynı zamanda, normal veya hastalıklı biyopsi doku il...

Video: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

İmmünoloji ve Enfeksiyon

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Yenidoğan Rat Beyin Dokusu Karışık Glial Hücre Kültürleri Primer mikroglia İzolasyonu

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Ölümsüzleştirilmiştir Sıralama B hücrelerinden Rekombinant İnsan IgG Monoklonal Antikorlarının Üretimi

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Hazırlık ve Organotipik Timik Dilim Kültürler Uygulamaları

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Fare Primer Hepatosit Kültürler bir inflamatuar yanıtın indüksiyon

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Yeni Nesil Electroporasyonun tarafından İlköğretim İnsan Th17 Hücreleri Küçük RNA Transfeksiyon

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...