This work was supported by the Swedish Cancer Society (11 0253, 13 0469), the Swedish Research Council (K2012-99X-21959-01-3), Marcus Borgstr&#246;ms Foundation and the Swedish Research Council through a grant to the Uppsala RNA Research Centre (2006-5038-36531-16). We are indebted to the BioVis core facility at Uppsala University for much help with the FACS analysis.

Protokol, insan hasta malzemeden lenfoid hücrelerin izolasyonu açıklar ve bu nedenle etik onayı gerekiyor. Bu çalışmada yapılan çalışmalar Uppsala Etik Değerlendirme Kurulu (DNR. 2013/387) tarafından verildi. İnsan Palatine Bademcikler dan Mononükleer Hücreleri (ÇUŞ) 1. İzolasyonu UYARI: kan, doku veya vücut sıvıları gibi insan kaynaklı tüm elenmemiş malzeme potansiyel enfekte materyal olarak kabul edilmelidir. Bu nedenle, insan dokuları işlemek için önerilen biyogüvenlik uygulamaları takip edilmelidir. Not: kontaminasyonunu önlemek için, her çözeltiler ve hücre kültürü ekipmanları steril olması gerekmektedir. Tüm tamponlar ve solüsyonlar önceden soğutulmuş ve buz üzerinde tutulmalıdır. Tonsil dokular ve izole hücreler tutulur ve buz üzerinde tutulmalıdır. <ol><li> Tonsillektomi veya tonsillotomy (Şekil 2) uygulanan hastalarda taze bademcikler edinin. Doku clum Plunge% 5 fetal sığır serumu (FBS), 10 mM Glutamin, 0,05 mg / gentamisin ve% 1 antibiyotik antimikotik karışımı ml (penisilin buz soğukluğunda steril Hanks ile dengelenmiş tuz çözeltisi (HBSS) ile dolu bir 50 ml&#39;lik santrifüj tüpüne ps Streptomisin ve Amfoterisin B). </li><li> Buz üzerinde her zaman batık bademcik dokusu örnekleri ile tüpleri tutun. En kısa sürede bademcik işlemek için çalışın, ve en geç cerrahi müdahaleden sonra 3 saat daha. </li><li> Buz üzerinde 60 mm plastik hücre kültür plaka üzerinde bademcik yerleştirin ve HBSS ile nemlendirilmiş doku tutun. Bademcik yüzeyinden temiz bir forseps ile görünür kan pıhtılarını, yağlı ve bağ dokuları çıkarın. </li><li> 5 ml HBSS içeren yeni 60 mm hücre kültürü plakası kullanın. Makas ve / veya bir neşter steril bir çifti kullanılarak 3-10 mm parçalara bademcik dokusu yığın kesin. </li><li> HBSS 10 ml içeren yeni 60 mm hücre kültürü tabak hazırlayın ve HBSS çözeltisi içinde 100 mikron plastik hücre süzgeç yerleştirin. </li><li> Diss aktarınSteril forseps ile hücre süzgecinden içine yansıtılmaktadır bademcik fragmanları. Plastik şırınga pistonu ucunu kullanarak, sorunsuz hücre süzgecinden doku parçaları sıkın. Bademcik fragmanları tamamen HBSS dalmış emin olun. </li><li> Doku kalıntıları hücre süzgecinden atın ve bir 50 ml plastik bir santrifüj tüpüne 60 mm hücre kültür plakasından elde edilen hücre süspansiyonu aktarın. Adımlar 1.8 ve 1.9 ile devam ederken buz üzerinde tüp bırakın. </li><li> Büyük bademcikler durumunda, boyutu 2 cm&#39;den büyük (Şekil 2), süzgeç örgü tıkanmasını önlemek için iki hücre süzgeçler de bademcik parçaları sıkın. </li><li> Hücre süspansiyonu 35 ml&#39;lik nihai hacim elde edilir. Not: Bu aşamada, dondurarak saklama (bakınız bölüm 2) hücre süspansiyonu hazırlamak mümkündür. </li><li> Yeni bir 50 ml santrifüj tüpüne Ficoll gibi yoğunluk gradyan çözeltisi 10 ml ekleyin. Yavaşça hücre Suspens bindirmeİyon yoğunluk gradyan çözeltisi üzerine (aşama 1,9). Hücre süspansiyonu ve yoğunluk dereceli çözeltisinin karıştırılması kaçının. Not: yoğunluk gradyan çözeltisi, oda sıcaklığına kadar ılıtıldı gerekir. </li><li> Oda sıcaklığında 20 dakika boyunca 700 xg&#39;de bir salıncak-out rotor hücre süspansiyonu santrifüjleyin. Degrade bozabilir hızlı frenleme olarak santrifüj fren işlevini etkinleştirmek etmeyin. Ayrıca, santrifüj mevcut olan en düşük ivme işlevini kullanın. Not: Bu santrifüj aşamasından sonra, tüpün alt kısmında tortu bir ara yüzeyde kabarık beyaz tabaka ve kırmızı kan hücrelerinin (RBC), fibroblastlar ve hücre kalıntıları olarak ÇUŞ&#39;larına dikkate alınmalıdır. </li><li> Dikkatlice 10 ml pipet kullanarak ÇUŞ&#39;ları katmanı toplamak. Yeni bir 50 ml santrifüj tüpüne hücre süspansiyonu yerleştirin. </li><li> Hücre süspansiyonuna buz soğukluğunda PBS 25 ml ilave edilir ve 4 ° C &#39;de dışarı salınan bir rotor 5 dakika boyunca 300 x g&#39;de tüp spin. </li><li> 25 ml&#39;lik bir pipet kullanarak süpernatantıve hücre pelet yıkama adımı iki kez daha tekrarlayın. Son olarak, PBS, 15 ml hücre pelletini. Not: Son yıkama aşamasından sonra, hücre süspansiyonu (bakınız bölüm 2) cryopreserve mümkündür. </li><li> Hemositometre hücre sayım bölmesi içinde hücre süspansiyonu 10 ul uygulayın ve bir mikroskop altında hücre sayısını. Hücreler uygun miktarda dağıtın (örneğin, 2-3 x 10 7) takip eden manyetik boncuk ayrılması için bir 15 ml&#39;lik santrifüj tüpüne. Adım 3.2 kadar buz üzerinde bu kısım tutun. </li></ol> Tonsil Hücre 2. Kryoprezervasyon <ol><li> Donma orta (% 90 FBS ve% 10 DMSO) hazırlayın ve buz üzerinde tutmak. Uzun süreli depolama için -20 ° C&#39;de donma medyayı tutmak. </li><li> Bir hemositometre hücre sayım odasını (adım 1.15) ile hücrelerin toplam sayısını belirler. İstenen donmuş hücre yoğunluğuna göre dondurma orta gerekli miktarını hesaplayın (örneğin, 10 7 hücres dondurma ortamı / ml). </li><li> 5 dakika boyunca 300 x g&#39;de hücre süspansiyonu santrifüjleyin. Hücre topaklarını bozmadan süpernatantı boşaltacaktır ve (aşama 2.1) buz gibi soğuk dondurma ortamı içinde hücre pelletini. </li><li> Sıvı nitrojen içinde uzun süreli depolama için tasarlanmış steril şişelere hücre süspansiyonu 1 ml&#39;lik numuneler koyun. Bir izopropanol odasında şişeleri dondurun ve -80 ° CO / N saklayın. Uzun süreli korunması için depolama tankı veya -140 ° C hücre dondurucu içeren bir sıvı azot içine şişeleri aktarın. </li><li> Dondurulmuş hücreler şişeleri çözülme için, 37 ° C su banyosu içinde hızla onları sıcak. Çözülmüş hemen zaman (takviyeleri ile 1.1 adım a) önceden ısıtılmış HBSS 10 ml hücre süspansiyonu dispers 15 ml santrifüj tüpüne. (1.1 adım takviyeleri ile), 5 dakika boyunca 300 x g&#39;de tüp Spin HBSS çıkarın ve HBSS, istenen hücre yoğunluğuna hücre pelletini. Not: ÇUŞ af canlılığınıter çözme Ölü hücrelerin varlığı saflaştınldı B ve T lenfositleri nihai verim düşecektir, önem taşımaktadır. Not: hücre izolasyon verimi azaltacaktır hücre canlılığı az% 80 daha sonra bizim deneyim göre. </li></ol> Tonsil ÇUŞ&#39;larının T Lenfosit Nüfus 3. Pozitif Seçimi Not 1: Bu protokol, CD3 antikoru bağlanmış manyetik boncuklar kullanılarak Tonsiller çok uluslu insan CD3 + T lenfositlerinin pozitif seçimi dayanmaktadır. Taze (Bölüm 1) veya dondurulmuş (Bölüm 2) çok uluslu şirketler bu bölümü başlatmak mümkündür. Not 2: 3 x 10 7 uluslu şirketler ile başlayın. Ayırma sütunları tıkayabilir ve bu izolasyon verimi azaltacaktır çünkü, bu hücre sayısı aşmayın. Daha fazla hücre ele alınacak olursa büyük sütunları kullanın. Bu protokolde kullanılan hacimleri deneysel hücrelerin bize sayısı için optimize edilmiştirBizim deneysel kurulum ed. <ol><li> Ayırma tamponu [PBS (pH 7.2),% 0.5 sığır serum albümini (BSA) ve 2 mM EDTA] hazırlayın. 4 ° C&#39;da bir 0.45 um filtre ve mağaza boyunca ayırma tamponu filtre. </li><li> 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de ÇUŞ&#39;ları süspansiyonu (basamak 1,14) dönerler. Buz soğukluğunda ayırma tamponu 240 ul ile sonuçlanan hücre pelletini (10 7 hücre başına 80 ul) yeniden süspanse edin. Steril 2 ml&#39;lik bir tüp içine hücre süspansiyonu aktarın. </li><li> Hücre çözeltisine, CD3 manyetik antikorun 20 ul ekle. Süspansiyon içinde bulunan hücrelerin durumda tutmak için sürekli hafifçe karıştırılarak, 4 ° C &#39;de 1 saat süre ile inkübe edin. </li><li> Hücreleri yıkamak için 5 mi, buzla soğutulmuş ayırma tamponu ihtiva eden 15 ml&#39;lik bir tüp içine hücre süspansiyonu her aktarın. </li><li> Dışarı salınan bir rotor, 4 ° C &#39;de 10 dakika boyunca 300 x g&#39;de santrifüjleyin tüp. </li><li> Bu arada manyetik ayırıcı ve sütun kurdu. Stand manyetik ayırıcı takın ve Separ sütun yerator. Buz soğukluğunda ayırma tamponu 500 ul uygulayarak sütun yıkayın. Akışını atın. </li><li> Sütunun altında yeni bir 15 ml toplama tüpü yerleştirin. Buz üzerinde toplama tüpü bulundurun. </li><li> Pipet ile süpernatantı (adım 3.5) atın ve yavaşça 500 ul ayırma tampon içinde hücre pelletini. Önceden yıkanmış sütunun üstüne hücre süspansiyonu uygulayın ve içinden çalıştırın. Not: Hücre kümeleri sütun yapışmasına neden olabilir ve bu şekilde akış hızını azaltabilir. Bu sorunu önlemek için, kolonun tepesinde bir 40 um plastik hücre süzgeci yerleştirin. Bu örgü sayesinde hücreleri Passing çok bu yöntemin etkinliğini artıracaktır. </li><li> Ayırma tamponu, 1.5 ml (10 7 hücreleri için 500 ul) ile sütun 4 kez yıkayın. Bir sonraki yıkama adımı uygulamadan önce (hiçbir sıvı sütununda dikkat edilmelidir) boş sütun rezervuar bekleyin. Not: B lenfosit kısmıyla olarak kabul CD3 etiketsiz hücreleri elde, oldukları gibitoplama tüpüne yıkandı. </li><li> Yeni 15 ml toplama tüpüne ayırıcı ve yerden sütun çıkarın. Pipet kolonuna ayırma tamponu 2 mi. T lenfosit fraksiyonu olarak kabul edilmektedir olumlu seçilmiş T lenfositleri, Zehir sütununda verilen piston kullanma. </li><li> Saflaştırılmış hücreler (adım 1.15) Gerekirse sayısını belirler. Hücre süspansiyonları, artık aşağı doğru deneyler için hazırdır. </li></ol> İzole 4. Akım Sitometri Analizi Tonsil B ve T Lenfositler DİKKAT: Paraformaldehit çözüm tahriş edici ve kanserojen olabilir. Çalışırken uygun koruyucu giysi, eldiven ve göz / yüz koruyucusu kullanın. Not 1: Bu protokol (FACS) analizi Kontrol THP izole edilmiş B ve T hücrelerinin doğrudan boyanması için bir yöntemi tarif etmektedir. Bu adımın amacı popülasyonları af izole hücre saflığını değerlendirmek içinter CD3 manyetik antikor ayırma. Bu amaçla kurulan B ve T hücre belirteçleri için fluorofor konjuge monoklonal antikorlar CD20-FITC ve CD2-APC kullanılır. Ayrıca izotip kontrol antikorları dahil yüksek CD2 ve CD20 antikorlarının (adım 4.8) arasında spesifik olmayan bağlanma &quot;arka plan&quot; ayırt önerilir. Not 2: Bu boyama zamanına kadar 4 ° C &#39;de bir% 1 paraformaldehid içinde karanlıkta (PFA) çözeltisi saflaştırılmış hücre fraksiyonları tutmak mümkündür (örneğin, ertesi gün).. Boyama ve FACS analizi arasında bir zaman boşluğu olduğu takdirde de aynı prosedür uygulanabilir. Her iki durumda da hücreler PBSA lekelenme veya FACS analizi öncesinde tamponu (PBS% 0.2 BSA ihtiva eden) ile düzgün yıkanmalıdır unutmayın. <ol><li> Adım 3.10 aşama 3.9 ve T lenfosit fraksiyonu, B lenfosit kısmını (önceki sütuna uygulamadan) aşama 1.15 den çok uluslu şirketler 6 x 10 6 hücre çıkar. </li><li> Hücreyi Spindışarı salınan bir rotor kullanılarak 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de süspansiyonlar. </li><li> Pipetle Süpernatantı ve buz gibi soğuk PBSA&#39;da 1 ml ile bir kez hücre pelet yıkayın. 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de hücre süspansiyonları dönerler. Pipetle Süpernatantı ve tekrar süspansiyon buz soğukluğunda PBSA tampon 600 ul hücre. </li><li> FACS tüpün alt hücre süspansiyonu 100 ul ekle. </li><li> PBS, her tüpe,% 10 ısı ile inaktive edilmiş insan serumu ihtiva eden 100 ul eklenir, iyice karıştırılır ve oda sıcaklığında ~ 1 dakika veya buz üzerinde 20 dakika inkübe edilir. Not: B lenfositleri Fc reseptörleri taşıyan. Fc bloke etmek için saflaştırılmış hücre fraksiyonları% 10 ısı ile inaktive edilmiş insan serumu ile inkübe edilir reseptörleri. Insan serumu, 1 saat boyunca 56 ° C&#39;de inkübe edilerek inaktive ısıdır. -20 ° C&#39;de dondurulur, küçük kısımlar ve mağaza, ısı ile inaktive edilmiş serum bölün. </li><li> Bir sw 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de santrifüj hücre süspansiyonuing-out rotor. </li><li> Pipetle Süpernatantı ve 1 ml PBSA&#39;da hücre pelet yıkayın. Santrifüj (4 ° C &#39;de 5 dakika süre ile 300 x g) tekrarlayın. </li><li> Buz üzerinde, her bir tüpe 100 ul PBSA ekleyin. Üreticinin tavsiyesine göre, florofor konjuge monoklonal antikorun uygun miktarda ekleme (örneğin, anti-CD20 20 ul ve / veya albümin 100 ul anti-CD2 antikorlarının 5 ul). Not: FACS analizi için yeterli kontrol tüpleri atamak unutmayın. Bu protokol, aşağıdaki kontroller dahil edilmelidir: ayrı ayrı anti-CD2 ve anti-CD20 antikorlar ile boyandı 1) hücreler, ek olarak bireysel olarak, anti-CD2 ve anti-CD20 izotip kontrol antikorları ve 3) hücreleri ile boyanmış 2) hücreleri Antikorların. </li><li> Kısaca tüp vorteks ve karanlıkta 4 ° C&#39;de 30 dakika inkübe edilir. </li><li> PBSA&#39;da 2 kez yıkayın. Örnekler 1,% PFA çözeltisi 2 ml ilave edilir. Hücreler 4 ° C&#39;de PFA çözeltide kalır edelim76; FACS analizi zaman kadar karanlıkta C. </li><li> Hemen FACS makinesinde örnekleri çalıştırmadan önce, (4 ° C&#39;de 5 dakika boyunca 300 x g), albümin ile hücreler 2 kez yıkayın. 1 ml PBS ile yeniden süspanse Numuneler akış sitometresi üzerinde ayırma, ışıktan korunan, 4 ° C&#39;de (ya da buz üzerinde) tutmak. </li><li> Üretici sitometresi akışından protokolü aşağıdaki FACS sıralama yapın. </li></ol> İzole Tonsil B ve T Lenfositlerindeki Adenovirüs DNA&#39;nın 5. PCR Algılama Not 1: adeno virüs hekson geni primerleri AdRJC1 (5&#39;-GACATGACTTTCGAGGTCGATCCCATGGA-3 &#39;) ve AdRJC2 (5&#39;-CCGGCTGAGAAGGGTGTGCGCAGGTA-3&#39;), 11. Bu primerler, 139 bp&#39;lik bir amplikon üretir. Ev sahibi 18S rRNA gen primerler tp206 (5&#39;-CCCCTCGATGCTCTTAGCTG-3 &#39;) ve tp207 (5&#39;-TCGTCTTCGAACCTCCGACT-3&#39;) ile tespit edilebilir. Beklenen Amplikon boyutu 300 bp olduğunu. Not 2: Her PCRÇalışma bir negatif kontrol (damıtılmış H2O) ve insan adenovirüs tip 5 12 ile enfekte edilmiş insan B hücre çizgisi (BJAB) elde edilen bir DNA pozitif kontrolü içerir. <ol><li> Silika membran bazlı kolon arıtma yöntemi kullanılarak (en az 1 x 10 6 hücre, adım 3.11) DNA ayıklayın. Fenol ve guanidin izotiyosiyanat çözeltisi 13 kullanarak, RNA yı ekstrakte edin. </li><li> 1x HF tamponu, deoksinükleotid trifosfat karışımı 0.2 mM (dNTP), 0.25, her bir primerden uM ve 20 ul bir toplam hacim içinde Yüksek Fidelity DNA polimeraz 1 U ihtiva eden bir reaksiyon karışımına, B ve T lenfositleri 100 ng DNA ekleyin. </li><li> Aşağıdaki çevrim koşulları altında PCR amplifikasyonu yapın: 10 sn, 67 ° C (hekson) ya da 58 ° C (18S rRNA) 30 sn için ve 72 ° C sıcaklıkta 98 ​​° C 30 döngü, ardından 98 ° C&#39;de 30 saniye denatürasyon, 30 sn. PCR reaksiyonu, 72 ° C&#39;de 7 dakikalık son bir uzatma aşaması ile sona erdirildi. </li><li> Elde edilen PCR amplif Ayrı1x TBE içinde% 1.5 agaroz jeli üzerinde ication ürünleri tampon ve nükleik asit boyası ile boyandı, beklenen şeritleri görselleştirmek. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+polymerase+chain+reaction+assay+for+rapid+diagnosis+of+conjunctivitis.">Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.</a> Invest Ophthalmol Vis Sci. 40, 90-95 (1999).</li><li>Zhang, Y., Huang, W., Ornelles, D. A., Gooding, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adenovirus+latency+in+human+lymphocyte+cell+lines.">Modeling adenovirus latency in human lymphocyte cell lines.</a> J Virol. 84, 8799-8810 (2010).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. biochem. 162, 156-159 (1987).</li><li>Watanabe, T., Yoshizaki, K., Yagura, T., Yamamura, Y. <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+antibody+formation+by+human+tonsil+lymphocytes.">In vitro antibody formation by human tonsil lymphocytes.</a> J Immunol. 113, 608-616 (1974).</li></ol>

The authors have nothing to disclose.

Bu protokolün sonucunu etkileyen en önemli faktörlerden biri başlangıç ​​malzemesi olarak taze tonsil malzeme kullanılmasıdır. Bu nedenle, bademcik örnekleri ameliyat sonrası 3 saat içinde işlenmelidir. Bademcikler yetişkinler hem de çocuklar elde edilebilir. Çocuklar bademcik malzeme nedeniyle bademcik (tonsillotomy) kısmi cerrahi olarak çıkarılması genellikle küçüktür. Bu nedenle, çok uluslu sayısı az tonsillektomi örneklerinde (1 x 10 8 -2 x 10 9) karşılaştır...

Tonsils are collections of incompletely encapsulated lymphoid tissues that lie under, and in contact with, the epithelium in the upper aero-digestive tract. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. The paired palatine tonsils together with the nasopharyngeal tonsil (adenoid), paired tubal tonsils and lingual tonsils constitute the so-called &#8220;Waldeyer&#180;s ring&#8221;. The latter is responsible for the initial contact between inhaled or ingested pathogens and the lymphoid tissues of the aerodigestive tract1,2. Indeed, numerous reports have shown that both bacterial and viral antigens can be detected in palatine tonsil tissue samples2-6.
The palatine tonsils are composed of dense lymphoid tissue covered by a stratified squamous non-keratinising epithelium. The tonsils have numerous crypts, epithelial invaginations, which penetrate the parenchyma increasing the surface area. Histologically, the palatine tonsils contain numerous lymphoid follicles with germinal centers, which are the sites for B cell maturation and differentiation (B-cell areas). Likewise, the palatine tonsils encompass T cells, which are mainly located in the extrafollicular regions (T-cell areas). In addition to the B and T cells, also various follicular dendritic cells can be detected in palatine tonsils1,2. 
Due to their anatomic location, the palatine tonsils are easily accessible by surgical interventions. For example, surgical removal of tonsils, known as tonsillectomy, is routinely carried out worldwide7. In children with tonsillar hyperplasia, a partial surgical removal of the tonsils (tonsillotomy) is sometimes used, causing less postoperative pain to the patients. Considering the accumulation of various pathogens in tonsils, surgically removed tonsils provide a unique opportunity to study the influence of viral and bacterial agents on tonsillar lymphocyte functions2,8. Furthermore it is possible to study if some pathogens prefer to reside in specific cell subpopulations9. In addition, as the tonsils are rich source of B lymphocytes, isolated tonsillar B lymphocytes can be efficiently used to study the activity of different B cell subpopulations10. However, as the palatine tonsils contain a mixture of cell types an efficient method to separate the different cell subpopulations is needed. 
Here, we describe a simple method for efficient and rapid isolation of tonsillar B and T cell populations from human palatine tonsils by using a magnetic-activated cell separation technique (Figure 1). The method described here is useful for scientists who want to assess the role of different infectious agents in human lymphoid organs such as palatine tonsils.

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			<td>Hanks balanced salt solution (HBSS)&#160;</td>
			<td>Gibco</td>
			<td>14175-053</td>
			<td>Contains 5% fetal bovine serum (FBS), 10 mM Glutamine,&#160;</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;0.05 mg/ml Gentamicin and 1% Antibiotic-Antimycotic mix</td>
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		<tr>
			<td>Freezing medium</td>
			<td></td>
			<td></td>
			<td>90% FBS and 10% DMSO</td>
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		<tr>
			<td>MACS buffer</td>
			<td></td>
			<td></td>
			<td>PBS (pH 7.2), 0.5% BSA and 2mM EDTA</td>
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		<tr>
			<td>PBSA&#160;</td>
			<td></td>
			<td></td>
			<td>PBS containing 0.2% BSA</td>
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			<td>PFA</td>
			<td></td>
			<td></td>
			<td>PBS containing 1% paraformaldehyde (PFA). Make fresh.</td>
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			<td></td>
			<td></td>
			<td></td>
			<td>PFA is suspected carcinogen. Wear gloves and goggles.</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Antibiotic-Antimycotic mix</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
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			<td>100-mm petridish</td>
			<td>Nunc</td>
			<td>172958</td>
			<td></td>
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			<td>Dissecting foreceps</td>
			<td>Fisher Scientific</td>
			<td>1381241</td>
			<td></td>
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			<td>Straight iris scissors&#160;</td>
			<td>Fisher Scientific</td>
			<td>12912055</td>
			<td></td>
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			<td>disposable scalpels</td>
			<td>Swann-Morton</td>
			<td>REF 0501</td>
			<td></td>
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		<tr>
			<td>100&#956;m plastic cell strainer</td>
			<td>Corning Life Sciences</td>
			<td>352360</td>
			<td></td>
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			<td>40&#956;m plastic cell strainers&#160;</td>
			<td>Corning Life Sciences</td>
			<td>352340</td>
			<td></td>
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		<tr>
			<td>2-ml plastic syringe</td>
			<td>BD Biosciences&#160;</td>
			<td>300185</td>
			<td></td>
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		<tr>
			<td>15-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62554502</td>
			<td></td>
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		<tr>
			<td>50-ml conical centrifuge tubes</td>
			<td>SARSTEDT</td>
			<td>62547254</td>
			<td></td>
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		<tr>
			<td>Low-speed centrifuge with fixed-angle or swinging-bucket rotor</td>
			<td colspan="2">Thermo Scientific Heraeus Megafuge 16R</td>
			<td></td>
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			<td>Ficoll&#8211;Hypaque</td>
			<td>Sigma-Aldrich</td>
			<td>F5415-50ML</td>
			<td>Ficoll solution</td>
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		<tr>
			<td>Fetal calf serum</td>
			<td>Biological industries</td>
			<td>040071A</td>
			<td></td>
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		<tr>
			<td>Dimethyl sulphoxide (DMSO)</td>
			<td>SIGMA</td>
			<td colspan="2">D2650-5X5ML</td>
		</tr>
		<tr>
			<td>MACS MS columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS human CD3 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-881</td>
			<td></td>
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			<td>MACS separator (Octo MACS)</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
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			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck Millipore</td>
			<td>1120180100</td>
			<td></td>
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		<tr>
			<td>EDTA</td>
			<td>AnalaR NORMAPUR</td>
			<td>20302.293</td>
			<td></td>
		</tr>
		<tr>
			<td>CD2 conjugated to allophycocyanin (CD2-APC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>560642</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20 conjugated to fluorescein isothiocyanate (CD20-FITC)&#160;</td>
			<td>BD Biosciences&#160;</td>
			<td>556632</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum&#160;</td>
			<td>Rockland Immunochemicals</td>
			<td>D119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-530L</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD Falcon</td>
			<td>352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72379</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRII flowcytometer</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva 4.1 software</td>
			<td>BD Biosciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Blood&#160;</td>
			<td>Macherey-Nagel</td>
			<td>740951.50</td>
			<td>DNA isolation kit</td>
		</tr>
		<tr>
			<td>TRIzol&#160; reagent</td>
			<td>Life technologies</td>
			<td>15596</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>The Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>0610030</td>
			<td>cell counter device</td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium</td>
			<td align="right">41003</td>
			<td>Nucleic acid gel stain&#160;</td>
		</tr>
	</tbody>
</table>

efficient isolation protocol for b and t lymphocytes from human palatine tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

B ve T lenfositlerinin yüksek oranda saflaştırılmış alt popülasyonlannda tonsil uluslu şirketlerin sonuçların verimli bir ayırma. Bu B ve T lemfosit popülasyonları sırasıyla (Şekil 3A) tespit etmek için bir anti-CD20 ve anti-CD2 antikorları kullanılarak FACS analizi ile teyit edilmiştir. Bunun aksine, B ve T lenfosit alt (Şekil 3B) hem de bir hücre karışımı hücre fraksiyonlarında uluslu şirketlerin sonuçların etkisiz bir ayrılması. <p class="jove_con...

Watch this Scientific Journal Video about Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils at JoVE.com

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

İnsan Palatine Bademcikler B ve T Lenfosit için Verimli İzolasyon Protokolü

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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15.7K Görüntüleme.  Uppsala Biomedical Center, Uppsala University. Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Video: Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Göç İnsan T Lenfosit İzolasyon, Kültür ve Analiz In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

İmmünoloji ve Enfeksiyon

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Kordon Kanı adlı Sitotoksik T lenfositler genişletilmesi olduğunu Hedef Sitomegalovirüs, Epstein-Barr Virus ve Adenovirüs

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Fare Genital Sistemlerinin Mukozanin gelen Lenfosit İzolasyonu

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Kordon Kanı gelen Prekürsör B-hücreli altkümeleri İzolasyonu

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo kullanılarak Elektron Cryotomography Rekonstrüksiyonunda ikinci viral zarf glikoproteini Spikes ortalamasını

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH ile Fare B Lenfositlerindeki kromozom anormallikleri hızlı Analizi

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Nucleofection İnsan THP-1 Makrofagların Yüksek Verimli Transfeksiyonu

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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, ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında