Yazarlar, Columbia Üniversitesi Mikrobiyoloji ve İmmünoloji Anabilim Dalı Dr. Boris Reizis yararlı tartışmalar için teşekkür etmek istiyorum. Ayrıca, akım sitometri ve video düzenleme ile ilgili yardım için Lu Huang ile teknik yardım için, Cornell Üniversitesi Veteriner Fakültesi Mikrobiyoloji ve İmmünoloji Anabilim Dalı Dr Margaret Bynoe, Dr. Rodman Getchell ve Deequa Mahamed teşekkür etmek istiyoruz. / - Fareler Yazarlar ayrıca p53 ıslahı ve genotipleme için kritik gözden geçirilmesi, el yazması ve video prodüksiyon ve Stephanie Yazinski Weiss laboratuvar Duhamel ve Weiss laboratuarlar üyelerine teşekkür etmek istiyorum. Cornell Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı. Bu araştırma, ABD Tarım Departmanı, Kooperatif Devlet Araştırma, Eğitim, ve Yayım Hizmetleri, Hayvan Sağlığı ve Hastalıkları Araştırma Programı (GED ve RSW) ve NIH hibe R03 HD058220 ve R01CA108773 (RSW) tarafından sağlanan fonları tarafından kısmen desteklenmiştir. Video Duhamel &amp; Weiss laboratuvarların personeli tarafından in-house imkanlarını kullanarak imal edilmiştir.

1. Tümör diseksiyonu <ol><li> Tümör diseksiyonu için, temiz bir kağıt havlu ya da tek kullanımlık cerrahi örtü diseksiyon pad üzerinde yerleştirilir ve% 70 etanol ile püskürtülür. </li><li> Fareler, bu protokol için rutin olarak CO 2 ile ötenazi ötenazi fare diseksiyon yastık yerleştirin ve etanol ile hayvanın her iki tarafta sprey . Bacakları içeri sokulur ve% 70 etanol ile iyice püskürtülür dört veya daha fazla itme pimleri kullanarak fare diseksiyonu ped üzerine bir yerde tutulur. </li><li> Steril makas kullanılarak altta yatan kasları kaçınarak ve vücut boşluklarında nüfuz ederken, orta hat üzerinde cilt pubis kafa tabanına kesilir. </li><li> Steril makas ve forseps iç organların zarar vermemeye dikkat ederek yeni bir çift ile karın boşluğuna açın. </li><li> Diseksiyon ve zararlı kan damarları olmadan ortaya çıkarmak için, tümörün göğüs kafesi saptırma ile göğüs boşluğu açın. </li><li> Diğer organlarda tümör ayırın ve soğuk PBS içine bir parça toplam tümör yaklaşık% 40-50 (yaklaşık 1.5 X 0.8 X 0.8 cm) transfer buz üzerinde tutulmalıdır. </li><li> Tümör kalan kısmı diğer deneyleri için kaydedilebilir ve tam bir nekropsi şu anda yapılabilir. </li></ol> 2. Kültürleme Lenfoma Hücreler <ol><li> % 70 etanol ve biyogüvenlik kabini transfer disseke tümör içeren tüp püskürtün. </li><li> Steril bir 150 mm 2 Petri kabı için 10 ml kültür ortamı ekleyin. </li><li> Petri kabı içine bir pipet ile hafif bir emme parça tümör aktarın. </li><li> Eğimli yan bakan iki steril 18 iğne bükün. </li><li> Bükülmüş iğneler kullanarak tümör ayrıştırmaları. </li><li> Swirl çanak hafifçe dokunun ve tümör büyük parça kaçınırken, bir yan hücre süspansiyonu toplamak için bir açıyla yerleştirin. </li><li> Tümörün büyük parça kaçınırken, 50 ml konik bir tüp içine hücre süspansiyonu ve transfer aspire edin. </li><li> Tümörün büyük parça kaçınırken, aynı tüp içine 10 ml orta ve transferi ile plaka yıkayın. </li><li> , Hafifçe lenfositler ve stromal hücrelerden oluşan hücre süspansiyonu karıştırın ve bir T-75 doku kültürü balona bir T-25 doku kültürü şişesi ve 10 ml 5 ml transfer. Matara yavaşça girdap. </li><li> 2.5 mL son hacmi (kültür damarlarının çeşitli türleri kullanarak farklı yoğunlukları kültür hücreleri T-hücre hattı kurulması olasılığını en üst düzeye çıkarır), 2, 4, 10.100 ve 1000 kat dilüsyonları yapmak için kalan hücre süspansiyonu kullanın. </li><li> 6 plaka bireysel kuyulara her seyreltme Levha 2.0 mL. Yavaşça girdap plakası. </li><li> Nemlendirilmiş CO 2 atmosferde hava hücreler 48 saat inkübe edin. </li></ol> 3. Besleme Lenfoma Hücreleri <ol><li> Inkübasyon 48 saat sonra hücrelerin minimal rahatsızlık ile duvar boyunca orta, T-75 balona T-25 orta şişeyi ve 10 ml 5 ml ekleyin. </li><li> 6 plaka hücrelerin minimal rahatsızlık ile duvar boyunca her iyi orta 2 mL ekleyin. </li><li> Ek bir 72 saat için hücreler inkübe </li></ol> 4. Lenfoma Hücreleri Pasajlanması <ol><li> İlk pasaj <ol><li> Inkübasyon beşinci günün sonunda orijinal şişeler ve levhalar (ilk geçişleri sırasında yaklaşık 3x106 / ml &#39;lik bir konsantrasyon hücre yoğunluğu korumak başarılı bir şekilde kurulması teşvik koruyarak, hücreler, 1:1 oranında geçişli. sürekli hücre hattı). <ol class="lalpha"><li> T-25 şişesi için 5 ml orta içeren yeni bir balona, ​​5 ml hücre süspansiyonu aktarın. Orijinal balona orta 5 ml ekleyin. </li><li> T-75 şişesi için, 10 ml orta içeren yeni bir balona 10 ml hücre süspansiyonu transfer. Orta orijinal balona 10 ml ekleyin. </li><li> 6 plaka için, her iyi orta 2 mL içeren yeni bir plaka ilgili kuyuların içine 2 ml hücre süspansiyonu her bir kuyudan aktarın. Orijinal kuyuların her bir kuyunun içine orta 2 mL ekleyin. </li></ol></li><li> Ek bir 72 saat boyunca tüm matara ve plakalar inkübe Bizim tecrübelerimize göre en hücre hatları bu geçit bir kültürler (gevşek yapışık süspansiyon hücre agrega varlığı ekli stromal hücreleri üzerinde kurulmuş bir hücre hattı erken bir göstergesidir) kurulmuştur. </li><li> Az ya da gevşek olarak yapışmış bir süspansiyon hücre agrega ile kültürleri için, dikkatli bir şekilde her 72 saat orta yaklaşık% 50 değiştirerek matara veya plakalar beslemek Orijinal kültür hücreleri kaldırarak önlemek için, kültür damarları supernatant orta hafif aspirasyon önce yerçekimi altındaki hücreleri tortu izin soluklu ve en az 5 dakika boyunca rahatsız edilmeden bırakılır. </li></ol></li><li> Kurulan hücre hatları bakımı <ol><li> Kurulan hücre hatları, yeni şişeler ve tabaklar içine geçişli. T-25 ve T-75 şişeler için, orijinal veya 5 ml ekleyingeçmesine orta 5 ml süspansiyon. 6-iyi plakalar için, T-25 balonuna orta 7 mL aslı veya geçit bir hücre süspansiyonu 3 mL ekleyin. Yem ve taze kültür ortamı ile temizlenebilir hacmi değiştirerek bir kültür aslı veya geçit korumak. </li><li> Passage, yeni T-25 şişeler içine hücre hatları kurdu. 18-24 saat hücre hatları iki katına zaman var Kültürleri rutin olarak 1-3x106 / ml yoğunlukta devam ve 3 gün her geçişli. Önceki pasaj kültürünün gerekli hacmi, 10 ml telafi etmek için yeni bir orta ekleyin. Yem ve özgün kültürünü korumak. </li><li> Birkaç pasajlar sonra, yapışmaz hücreler kendini yapışık stromal hücreler olmadan sürdürmek ve yapışık hücrelere daha küçük bir sayı her geçiş yapılır. Bu noktanın ötesinde, kültürleri, T-25 şişeler tedavi olmayan doku kültürü muhafaza edilebilir. </li></ol></li></ol> 5. Lenfoma Hücre Hatları dondurulması ve Kurtarma <ol><li> Hücre hatları% 20 FBS ve% 10 DMSO içeren taze hazırlanmış RPMI 1640 orta dondurulur. </li><li> T-25 balonuna (yaklaşık 1.5x10 6 hücre / ml) 48 saat eski kültür Hücreler oda sıcaklığında 5 dakika 1500 rpm&#39;de santrifüj. Atarak süpernatantı sonra, 2 mL soğuk dondurma orta hücrelerin yeniden askıya alınır. Her balona iki hücre süspansiyonu 1.0 mL hacimli hemen -80 dondurulmuş, donma şişeleri yerleştirilir ° C, ve ertesi gün sıvı azot aktarılır. </li><li> Hücreler, hızlı bir çözülme ve 10 ml taze kültür ortamı içindekilerin resuspending sıvı azot depolama kurtarıldı. Alternatif olarak, 10 ml taze kültür ortamı resuspending önce DMSO kaldırmak için taze çözülmüş hücreler ılık kültür ortamı kez yıkanır. Hücreleri sonraki geçirilmesinden önce en az 72 saat süreyle kültüre. </li></ol> 6. Temsilcisi Sonuçlar Bir Atm / - T-hücre hattı bu protokolü kullanılarak geliştirilen ve belirlenmiş DWJ-Atm-1 anti-CD3-FITC, anti-CD4 APC ve anti-CD8-PE ve akış sitometri analizi (Şekil 1) ile boyama ile karakterize edilmiştir. T-hücrelerinin% 90&#39;dan fazlası, CD3, CD4 ve CD8 triple-pozitif. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Şekil 1" /> Şekil 1 bu protokolü kullanarak geliştirilen T-hücre hattı Karakterizasyonu. / - - Kurulmuş bir Atm hücre yüzey belirteçleri hücre hattı DWJ-Atm-1 ile boyanması ile karakterize edilmiştir atanmış anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE ve flow sitometri analizi. Kısaca, 1x10 6 taze hücreler soğuk PBS (1500 d / 5 dk) bir kez yıkanmış ve 4 inkübe ° C, PBS içinde% 1 BSA ile 1:200 dilüsyon antikor kokteyli (her antikor mcL 200 ile 10 dk . taze veya daha sonra analiz için sabit. Sabit hücreleri bir final konsantrasyon% 4 (hacim / hacim) ekleyerek nötr tamponlu formalin tarafından hazırlanan ve 30 dakika santrifüj yoluyla hasat, (4 ° C&#39;de inkübe ise hücreler hemen incelenebilir 1500 d / 1 X 10 4 hücrelerinin toplam 5 dakika) ve% 1 BSA ile PBS 200 mcL yeniden bekletildi. BD FACS Calibur analizörü ve CellQuest yazılımı (BD Biosciences, San Jose, CA) kullanılarak analiz edildi.

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

Bu protokolde, iki farklı fare genotipleri fare T-hücre hatları kurmak için ayrıntılı yordamlar sağlar; - / - [FVB/N- Atm tm1Led / J] ve p53 - / - Atm [129/S6- Trp53 tm1Tyj / J] . / - Ve üç (5 girişimleri), p53 - / - Bu protokol, 6 olmak üzere toplam (7 girişimleri) Atm kullanarak fare T-hücre laboratuvarda kurulmuştur. / - Bu hücre hattı DWJ-Atm-1, bir <em...

<ul><li> Buz kovası </li><li> % 70 etanol </li><li> Alüminyum folyo ve pimler ile kaplı köpük blok oluşan Diseksiyon ped </li><li> İki takım küçük steril makas ve forseps </li><li> Steril 150 mm 2 Petri kabı (Thermo Fisher Scientific, Waltham, MA, kedi # 08-757-13) </li><li> Steril 18 insülin iğnesi </li><li> 50 ml konik tüpler (Corning Inc Corning, NY, kedi # 430.829) </li><li> % 10 formalin solüsyonu </li><li> Doku kültürü matara <ul><li> T-25 (DYP, Trasadingen, İsviçre; kedi # 90025.) </li><li> T-75 (DYP, Trasadingen, İsviçre; kedi # 90.075) </li></ul></li><li> 6-kuyucuğu (Corning Inc, Corning, NY, kedi # 3506) </li><li> Steril fosfat tuzlu pH 7,2 (PBS) tamponlu </li><li> Kültür ortamı <ul><li> L-glutamin (Lonza, Walkersville, MD; kedi # 12-115F) 200 mM, 25 mM HEPES RPMI 1640 aşağıdaki ile desteklenebilir: <ul><li> Inaktive% 10 ısı (56 ° C, 30 dakika) fetal sığır serumu (FBS; Hyclone Logan, UT, kedi # SH30088.03) </li><li> % 1 penisilin ve streptomisin (Mediatech, Manassas, VA, kedi # 30-002-CI) </li><li> % 1 aminoasitlerin (Mediatech; kedi # 25-025-CI) </li><li> 55 mM 2β merkaptoetanol (Sigma, St Louis, MO, kedi # M752) </li></ul></li></ul></li><li> Dimetil sülfoksit (DMSO; Calbiochem, La Jolla, CA, kedi # 317.275) </li><li> Antikorlar (eBioScience, San Diego, CA) <ul><li> anti-CD3-FITC (kat. no 11-0031-85) </li><li> anti-CD4-APC (kat. no 17-0041-83) </li><li> anti-CD8-PE (kat. # 12-081-85) </li></ul></li><li> Sığır serum albumin (BSA; Yaşam Teknolojileri, Grand Island, NY, kedi # 11.018-017) </li><li> Concanavalin A (Sigma kedi # C5275) </li><li> 1.8 mL dondurma şişeleri (Nunc, Roskilde, Danimarka, kedi # 368.632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Kurulan hücre hatları araştırma laboratuvar hayvanları kullanımını azaltmak kritik bir araştırma aracı. ATM gibi genetiği değiştirilmiş farelerde Bazı suşları / - ve p53 - / - sürekli erken ömrü 1,2 timik lenfoma geliştirmek, ve böylece, fare T-hücre hatları türetilmesi için güvenilir bir kaynak olarak hizmet verebilir. Burada gerek kalmadan önceki protokoller 1,3 açıklandığı gibi interlökinler eklemek için kurulan fare timik lenfoma T-hücre gelişimi için ayrıntılı bir protokol mevcut. Tümörler, hunched duruş gözlem dayalı görünür tümörlerin erken göstergesi, üç ila altı ay arası farelerden alınan hasat nefes alma, yetersiz bakım ve duyarlı bir gerginlik 1,4 de israf yorucu. / - [FVB/N- Atm tm1Led / J] 2 ve p53 - / - [129/S6- Trp53 tm1Tyj / J] 5 fareler Biz bu protokolü kullanarak çeşitli T-hücre hatları ve Atm kendilenmiş suşları başarıyla kurduk. Daha kurulan T-hücre nüfusunun% 90&#39;dan daha fazla CD3, CD4 ve CD8 ifade ettiğini göstermektedir. Stably kurulan hücre hatları ile tutarlı, mevcut protokolü kullanılarak üretilen T-hücrelerinin, bir yılı aşkın bir süredir geçişli olmuştur.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Bu video, fare timik lenfoma hücre hatları kurmak için bir protokol göstermektedir. Ve p53-/ - farelerde timus lenfoma ile bu protokolü takip ederek, başarıyla birkaç Atm / T-hücre hatları kurduk.

Timik Lenfoma T-hücre Hatları türetilmesi Atm - / -</sup Ve P53 - / -</sup Fare

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K Görüntüleme.  Cornell University. Bu video, fare timik lenfoma hücre hatları kurmak için bir protokol göstermektedir. Ve p53-/ - farelerde timus lenfoma ile bu protokolü takip ederek, başarıyla birkaç Atm / T-hücre hatları kurduk.

Video: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Karşı Önceden tanımlanmış T Hücre Reseptör özgüllükleri Transnuclear Fare Toxoplasma gondii SCNT ile Elde Edilen

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

İmmünoloji ve Enfeksiyon

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Akım Sitometri tarafından Timik Pozitif ve Negatif Seçim incelenmesi

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T hücrelerinin türetilmesi<em&gt; İn Vitro</em&gt; Fare Embriyonik Kök Hücreleri

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Kemik iliği projenitör hücrelerinin retroviral İletimi T-hücre reseptör Retrogenic fareler üretmek için

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK ve kronik aktif Epstein - Barr virüsü enfeksiyonu olan hastalarda T hücre satırlarından kurmak için etkili ve basit bir yöntem

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Virüslü Ifnar1- / - farelerin Immunoprivileged organlarında Zika virüs özgü T hücreli yanıt-e doğru değerlendirilmesi

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

MACROPHAGE ve T Hücre Hatlarında CRISPR/Cas9 ve Hücre Sıralama tarafından Gen Knock-in

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Timik Lenfoma T-hücre Hatları türetilmesi Atm - / - Ve P53 - / - Fare

Timik Lenfoma T-hücre Hatları türetilmesi Atm^{- / - Ve P53^{- / - Fare}}