Name: isolation and transplantation of different aged murine thymic grafts.
Uploaded: 2015-05-13T14:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

This work was supported by funding received from the National Institutes of Health (1R01AI083269).

kemirgen çalışmalar Kuzey Carolina Chapel Hill Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmış ve tüm hayvan bakımı IACUC kurallarına uygun olarak yapıldı. Yenidoğan ve Erişkin Thymi 1. Hazırlanması <ol><li> Önce donör fareler euthanizing tüm reaktifler ve donanımları hazırlayın. </li><li> Otoklav veya diğer uygun yöntemlerle cerrahi aletler sterilize edin. Tüm cerrahi prosedürler, steril koşullar korumak ve kirlenmeyi önlemek için bir laminar akış başlığı altında gerçekleştirilmelidir. Donör farelerden alınan Thymi çıkarılması için gerekli araçları birleştirin. </li><li> Steril 1x PBS (pH 7.4) 60 mm çanak doldurun ve başlık içindeki buz üzerine yerleştirin. Bu transplantasyondan önce donör farelerden kesilmiş kısa deposu Thymi için kullanılacaktır. </li><li> Etik kurallar gereğince, CO servikal dislokasyon tarafından takip 2 boğulma decapitation ve yetişkin donör fareler tarafından yenidoğan donör fareler euthanize. </li><li> Rtimus emoval: Steril bir emici kağıt havlu üzerine dorsal yaslanmış pozisyonda fare Lay ve bir kesi yapmadan önce% 70 etanol ile sprey. </li><li> Deri yoluyla orta hat kesi yaparak karın ve göğüs boşluğu Açığa. Göğüs boşluğu ortaya çıkarmak için göğüs ve ön ayakları üzerinde cildi katlayın. </li><li> Üstün mediasten ortaya çıkarmak ve göğüs boşluğu anterior diyafram ve göğüs kafesi üzerinden iki yanal kesiler olun. timus hemen üstünde iki beyaz lob olarak görünür ve kalp bitişik olmalıdır. </li><li> Kapsülü bozmak için değil bazı yapım, ince forseps ile timus çevreleyen bağ dokusu dışında tease. Forseps ile göğüs kafesi geri tutarken, organ altında kavisli bir forseps konumlandırma ve dikey çekerek timus iki lobunun ayıklamak için forseps ikinci bir çift kullanın. Bu yeni doğmuş farelerden Thymi ayrılması için bir kesme kapsamı kullanılarak yapılabilir. </li><li> 60 mm di timus yerleştirinsh bağ berzah ile keserek buz ve ayrı timik lobları üzerinde steril 1x PBS (pH 7.4) içeren. Kapsül zarar vermemek için bazı yapım timik lob herhangi enkaz kaldırmak ve transplantasyon için bölümlere uygun sayıda içine timus kesti. </li><li> Yeni doğmuş farelerden elde edilen Thymi manipüle etmeyin. NOT: İki alıcı farelere (alıcı başına 1 lob) en fazla kullanılır. </li><li> 4-6 alıcı farelerin maksimum olarak, yetişkin farelerden elde edilen Thymi nakli. Şekil 1&#39;de gösterildiği gibi dikkatle, bir yetişkin timik lobunu kavramak, forseps bir çift kullanarak, cerrahi makas kullanarak üç eşit parçaya timus kesti. </li><li> Yineleyin her donör fare için 1.3-1.4 adımları. Maruz Thymi zamanı sınırlamak amacıyla, bir defada 1 donör timus hazırlar. </li></ol> Böbrek Kapsül altında 2. Thymus İmplantasyonu <ol><li> Tablo 1 &#39;de listelenen önceden sterilize edilmiş donanım bir araya getirin. Uygun aseptikteknik, kontamine araç veya reaktif transplant-alıcıları açığa önlemek için prosedür sırasında kullanılmalıdır. </li><li> Transplantasyon öncesinde, tartmak ve her alıcı fare etiketleyin. </li><li> Laminer akış kaputu diseksiyon mikroskobu ve anestezi devresi ayarlayın. </li><li> Bir elektrik ustura kullanarak, alıcı farenin sol tarafını tıraş ve hiçbir saç kesi yapmak için kullanılan alanı çevresinde kalmasını sağlamak. </li><li> Isofluorane buharlaştırıcı açın ve 1-2 arasındaki% bir doz kullanılarak fare uyutmak. Önce ayak tutam aşağıdaki refleksinin yokluğu kontrol ederek cerrahi prosedürü başlamadan uygun anesthetization tespit. </li><li> Fare düzgün uyuşturulduktan sonra, kuruluk önlemek ve şöyle nakli için fareyi hazırlamak için fare gözlerine veteriner merhem uygulayın: </li><li> Traş yüzü yukarı bakacak şekilde sağ yanal yaslanmış pozisyonda mikroskop altında fare yerleştirin. Starting cerrahi alanın merkezinde, povidon-iyot (betadin) takip atılan bir aktarım pipet% 70 etanol kullanarak dairesel hareketlerle dağıtmak. Bir kesi yapmadan önce 3 kez etanol / betadine tedavi tekrarlayın. </li><li> Kullanımı diseksiyon makas doğrudan böbrek üstünde 1-2 cm kanat kesi yapmak. </li><li> Böbrek bitişik bağ dokusu kavramak ve kas üstüne yatıyor şekilde hafifçe kaldırın böbreği orta forseps kullanarak. Kas üzerinde maruz böbrek ve yerinde tutmak için, böbrek iliyum bozmaya değil özel dikkat, böbrek altında orta forseps bir kolunu yerleştirin. </li><li> Doku kurumayı önlemek amacıyla, böbrek ve böbrek kapsülü manipülasyonu tamamlanana kadar her adım sırasında steril 1 x PBS (pH 7.4) böbrek yıkayın. </li><li> Ince forseps tutam ve böbrek ayırmak için böbreküstü bezlerinin en uzak böbrek kenarına yakın kapsülü kaldırın kullanma. </li><li> 18 ölçeri kullanınİğne donör timus (Şekil 2A) Hazırlanan parça eklemek için yeterince büyük bir kesi yapmak. Zaman içinde greft çıkık önlemek için mümkün olduğu kadar küçük kapsüler kesi tutmak için emin olun. </li><li> Kapsülü tutarak böbrek uzağa çekti ederken, kapsül altında greft yerleştirin ve mümkün (Şekil 2B) olarak kapsül kesiden kadar ileri timus itmek için ince forseps ikinci bir çift kullanın. </li><li> Böbrek altından forseps kaldırın ve yavaşça kesiden yerine geri böbrek dönün. </li><li> Periton duvarını dikilmesi ve bir kez kapalı betadin uygulayarak kas kapatın. Dikiş tamamlandıktan sonra, dermis kapatın ve çevredeki betadin uygulamak için yara klipleri geçerlidir. </li><li> Bir ısı lambası kullanarak ısındı edilmiş bir kafes fare dönün. </li><li> Tam bilinç ve hareketlilik kavuşur kadar her post-operatif fare izleyin ve fareler tha koymayınt diğer hayvanlarla bir kafesin içine anestezi aşağıdaki kurtarıldı değil. </li><li> Gerektiği gibi post-operatif ağrı yönetimi ilaçlarla fareler davranın. Örneğin 1.6 mg / ml&#39;lik bir konsantrasyonda, içme suyu içinde asetaminofen gibi ameliyat sonrası analjezikler ile fareler sağlar. </li><li> Yineleyin her alıcı fare için 2,3-2,9 adımları. </li><li> 1 saat sonrası ameliyat içinde normal aktivitesine bu fareler dönüşünü sağlayın. Nakli prosedürünün bir sonucu olarak ortaya çıkan komplikasyonları belirlemek için post-operatif ağırlık, motor becerileri, yanı sıra, her fare içme ve beslenme etkinliğini izlemek. NOT: pre-operatif ağırlık yanı sıra değişmiş hareketlilik ya da yemek ya da içmek komplikasyonları gösterebilir başarısızlık kıyasla Ameliyat sonrası kilo kaybı,. </li><li> Kuyruk nick üreticinin talimatlarına göre limpholite hücre ayırma medyayı kullanarak lenfositlerin ayrılması ardından yoluyla farelerin kanama ile periferik kan T hücre sulandırılması izleyin. T hücresi characteriza başarmakBöyle bir floresan aktive sukkinimidil ester 8 gibi T hücre belirteçleri, CD3, CD4, CD8, hem de canlı ölü diskriminatör özgü floresan konjuge antikor ile lenfositlerin boyanarak tion. Şekil 3 &#39;de gösterildiği gibi analiz tekli, canlı hücreler ve CD3 pozitif hücreler üzerinde geçiş yoluyla gerçekleştirilebilir. </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.+Immunophenotyping.">Chapter 6. Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Rice, H. E., 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=Thymic+transplantation+for+complete+DiGeorge+syndrome:+medical+and+surgical+considerations.">Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations.</a> J. Pediatr. Surg. 39 (11), 1607-1615 (2004).</li><li>Markert, M. L., 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=Transplantation+of+thymus+tissue+in+complete+DiGeorge+Syndrome.">Transplantation of thymus tissue in complete DiGeorge Syndrome.</a> N. Engl. J. 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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

Timus T hücre gelişiminde kritik olaylar 1 meydana geldiği bir organdır. Yerleşik timositleri, T hücresi reseptörü (TCR) α ve β genlerin tekrar düzenlenmesi üzerine timus 2 kortikal ve medüller bölgelerde lymphostromal ve antijen sunan hücreler (APC) ile etkileşimlerin, bir dizi geçer. Timus pozitif seçim ana majör histokompatibilite kompleksi (MHC) sınıf I ve II moleküllerinin 2-3 bağlamında antijenik peptitleri tanıyan timositleri üretilmesi için kortikal timik epitelyum hücreleri (TEC) aracılık eder. Daha sonra timik negatif seçim medüller TEC veya dendritik hücrelerin (DC), MHC sınıf I ve II moleküllerinin 3 ile bağlanmış kendi proteinlerinden türetilen bu, mevcut peptidler ile bir etkileşim ile tahrik edilen reaktif timositlerin tasfiye gerektirir. Bu işlemlerin bir sonucudur geniş bir yelpazede cevap verebilmek olgun CD4 + ve CD8 + T hücrelerinin bir havuz kurulmasıyabancı antijenlere kendi kendine proteinlerin 4 minimal reaktiviteye sahip iken. timik seçim olayları verimliliği timus olgunlaşması, meduller ve kortikal TEC, timik DC alt kümesi bileşiminin sıklığı ve timik öncüleri 3 kaynağı gibi faktörlerin, bir dizi etkilenir. Özellikle, anormal timik seçim sırasıyla, bozulmuş negatif veya pozitif seçimden kaynaklanan otoimmun 5 veya immünyetmezligi patolojiler, neden olabilir. timik seçimi düzenleyen moleküler olaylar, ancak tam olarak anlaşılamamıştır. In vitro böyle reaggregate timik organ kültürleri (RTOC) 6 olarak yaklaşımları, timik seçimi ile ilgili temel olayları analiz etmek için yararlı olabilir, ancak tam sürmekte dinamiklerini recapitulate başarısız kanıtlanmıştır in vivo olaylarda. Sonuç olarak, bu timik transplantasyon dayalı yaklaşım, in vivo 7 daha iyi çalışma timosit seçim olayları için kurulmuştur </s&gt; yedekleyin. Bu protokol bağışık yetmezliği scid alıcı farelere yenidoğan ve yetişkin donör farelerin Thymi nakli açıklanır. Bu teknik, pozitif ve negatif timik seçimi, hem de ontogeny sırasında çeşitli T hücresi alt kümelerinin timik çıkış düzenleyen mekanizmaların çalışmasına izin verir. En son olarak, bu yaklaşım, timik Seçimin verimi, erken otoreaktif T hücrelerinin artan gelişmesine yol açan, farelerde doğumdan sonra sınırlı olduğunu göstermek için kullanılır ve yabancı antijenlere 7 için azaltılmış T hücresi repertuarı spesifik edilmiştir.

<table><tbody><tr><td>Ethanol&amp;nbsp;</td><td>Decon Laboratories</td><td>2705HC</td><td></td></tr><tr><td>PBS</td><td>GIBCO</td><td>14190-144</td><td>pH 7.4</td></tr><tr><td>Betadine Solution</td><td>Purdue Products</td><td>67618-150-17</td><td></td></tr><tr><td>18 gauge needle</td><td>BD</td><td>305195</td><td></td></tr><tr><td>Sutures (1.0 metric)</td><td>Ethicon</td><td>J493G</td><td>18&quot; (45cm)</td></tr><tr><td>AUTOCLIP Wound Clips (9mm)</td><td>Clay Adams&amp;reg; Brand</td><td>427631</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher Scientific</td><td>13-711-20</td><td>Sterile, disposable</td></tr><tr><td>Mouse anti-CD3 Ab</td><td>eBioscience</td><td>11-0031-85</td><td>Clone: 1452C11</td></tr><tr><td>Mouse anti-CD4 Ab</td><td>eBioscience</td><td>48-0042-82</td><td>Clone: RM4-5</td></tr><tr><td>Mouse anti-CD8 Ab</td><td>eBioscience</td><td>25-0081-82</td><td>Clone: 53-6.7</td></tr><tr><td>Lancet</td><td>Medipoint</td><td>Goldenrod 4mm</td><td></td></tr><tr><td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td><td>Invitrogen</td><td>P30253</td><td></td></tr><tr><td>96-well round botton polypropylene plates</td><td>Corning</td><td>3365</td><td></td></tr><tr><td>1.2 ml polypropylene cluster tubes</td><td>Corning</td><td>4401</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD</td><td>352002</td><td></td></tr><tr><td>40uM Nylon Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>16% Paraformaldehyde Solution, EM Grade</td><td>Electron Microscopy Services</td><td>15710</td><td>Hazardous</td></tr><tr><td>Puralube Optical Ointment</td><td>Fisher Scientific</td><td>NC0138063</td><td></td></tr><tr><td>Lympholyte</td><td>Cedarlane</td><td>CL5030</td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Corning</td><td>430196</td><td>60 mm x 15 mm, Sterile</td></tr></tbody></table>

isolation and transplantation of different aged murine thymic grafts.

timik seçim etkinliğini düzenleyen mekanizmalar kötü tanımlanmış kalır. in vivo kendi kendine ve yabancı antijenleri için spesifik T hücrelerinin gelişmesine ve seçim analizleri burada sunulan yöntem sağlar. yaklaşım immünyetmezligi scid alıcıların içine çeşitli yaşlı farelerde türetilen timik greft implantasyonu gerektirir. Süresi nispeten kısa bir süre boyunca alıcı tam implante timus greft türetilen T hücreleri ile yeniden oluşturulmuştur. Sadece timositler, izolasyon anında timus tohum seçimi geçmesi ve olgun T hücreleri dönüşür. Bu nedenle, yaşa bağlı timik olayların bir fonksiyonu olarak aşılanan T hücrelerinin yapısı ve özgüllük değişiklikler değerlendirilebilir. Teknik uzmanlık başarılı timik transplantasyonu için gerekli olsa da, bu yöntem, in vivo olarak anormal timik fonksiyonu ve / veya homeostas sonucunda ya da bağlı olan patolojilerin geniş bir çalışma için benzersiz bir strateji sağlarolduğunu.

Bu prosedürün başarısı az cerrahi travma gibi böbrek kapsülü altından greft doğru konumlandırma bağlıdır. Şekil 1 &#39;de gösterildiği gibi, timik greft uygun temin müteakip transplantasyon için timik bölümleri boyutlu şekilde kesilmelidir. Şekil 1&#39; de şematik sonra, yeni doğan veya yetişkin farelerden Thymi tutarlı ve tekrarlanabilir bir T hücre nakli sonuçları ile başarılı bir subkapsüler nakli için de kullanılabilir. Belirtildiği gibi, böbrek...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

İzolasyon ve Farklı Yaşlı Fare Timik Greft Nakli.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.7K Görüntüleme.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

Video: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Timus Karakterizasyonu kullanma Fare Embriyo atalarıdır Settling<em&gt; In Vivo</em&gt; Ve<em&gt; In Vitro</em&gt; Tahliller

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Gelişim Biyolojisi

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2-Foton Mikroskopi kullanarak Fare Thymus intravital Görüntüleme

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

İmmünoloji ve Enfeksiyon

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host hastalığı indüksiyonu ve<em&gt; In Vivo</emBir MHC-eşlemeli murin Model Kullanma&gt; T Hücre İzleme

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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 ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Mürin Tam kat Cilt Nakli

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Farelerde A Novel Mikrocerrahi Heterotopik Modeli, En Bloku Göğüs Duvarı, Thymus ve Kalp Nakli

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Tıp

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

Çıplak farelerde 2 '-Deoxyguanosine tedavi Murine embriyonik Thymus renal subkapsüler nakli

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Timus ve Kemik İliğinden Stromal Hücre İzolasyonunun Optimize Edilmesi

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Yazar Spotlight: Tek Hücre Dizilimi için Stromal Hücre İzolasyonu Kullanarak Hematopoietik Araştırmaları İlerletmek

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

İzolasyon ve Farklı Yaşlı Fare Timik Greft Nakli.

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Bu videodaki bölümler

Timus Karakterizasyonu kullanma Fare Embriyo atalarıdır Settling

2-Foton Mikroskopi kullanarak Fare Thymus intravital Görüntüleme

Graft-versus-host hastalığı indüksiyonu ve

Farelerde allojenik CD4 + T hücre Cevapları Eğitim için Kuyruk Cilt Nakli

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

Mürin Tam kat Cilt Nakli

Farelerde A Novel Mikrocerrahi Heterotopik Modeli, En Bloku Göğüs Duvarı, Thymus ve Kalp Nakli

Çıplak farelerde 2 '-Deoxyguanosine tedavi Murine embriyonik Thymus renal subkapsüler nakli

Hematopoetik Organlardan Stromal Hücre İzolasyonu

Hematopoetik Organlardan Stromal Hücre İzolasyonu