Çalışma Eğitim, Singapur Bakanlığı (AcRF Tier1-RG40 / 13 ve Tier2-MOE2013-T2-2-038) tarafından desteklenmektedir. el yazması Obrizus Communications Amy Sullivan tarafından düzenlendi.

Tüm fareler yetiştirilen ve özel patojen içermeyen koşullar altında korunur ve tüm fare protokolleri Kurumsal Hayvan Bakım ve Kullanım Komitesi kurallarına uygun olarak yürütülmektedir vardır. Tamponlar ve Reaktiflerin hazırlanması 1. <ol><li> 55 uM 2-merkaptoetanol tamamlandı Roswell Park Memorial Institute (RPMI) ortamında (% 10 ısı ile inaktive edilmiş fetal büyükbaş hayvan serumu (FBS), 2 mM L-glutamin, penisilin (100 lU / ml) / streptomisin (100 ug / ml) hazırlayın ). </li><li> Ayrı, 20x Dengeli Tuz Çözeltisi (BSS) Stok 1 ve Stok 2 hazırlayın. <ol><li> 20x BSS stok 1 (1 L steril su içinde 111 mM dekstroz, 8.8 mM potasyum fosfat, 26.7 mM sodyum fosfat dibazik) hazırlanması ve son hacim ayarlamasından önce, 20x BSS stoklar 1-40 ml% 0.5 Fenol Kırmızısı ekleyin. 4 ° C&#39;de depolamadan önce 0.2 mikron filtre kullanılarak steril filtre. </li><li> 20x BSS stok 2 (25.8 mM kalsiyum klorür dihidrat, 107 mM potasyum klorür, 2.73 M sodyum ch hazırlayınloride, 19.6 mM magnezyum klorür hekzahidrat, 1 L&#39;lik steril su içinde 16.6 mM magnezyum sülfat). 4 ° C&#39;de depolamadan önce 0.2 mikron filtre kullanılarak steril filtre. </li><li> 400 ml steril su her ayrı 50 ml BSS stok 1 ve 50 ml BSS stok 2 sulandırmak, deneysel kullanım için 1x BSS hazırlamak. , Seyreltilmiş çözüm birleştirin pH 7&#39;ye ayarlamak ve 20 ml FBS (% 2) ilave edilir. 0.2 um filtre kullanılarak steril su ve steril filtre kullanılarak 1 L doldurun. NOT: Konsantre BSS stoklarının karıştırılması doğrudan yağış neden olabilir çünkü BSS stok çözeltileri ayrı ayrı hazırlanmalıdır. </li></ol></li><li> Kırmızı kan hücresi (RBC) Liziz Tamponu <ol><li> RBC liziz tamponu kullanımdan önce 1 parçası hazır Tris-baz (steril su içinde 130 mM Tris (hidroksimetil) aminometan, pH 7.65) 9 parça hazır amonyum klorür (steril su içinde 155 mM amonyum klorür) karıştırın. Not: 4 ° C&#39;de amonyum klorür ve Tris-bazın mağazası steril stok çözeltileri. lizis b hazırlayınRBCs verimli parçalama sağlamak için taze Uffer. </li></ol></li></ol> Dalak veya lenf nodu gelen Lenfosit Süspansiyon 2. Nesil NOT: fare ötanazi önce deney için gerekli tüm reaktifler ve ekipman hazırlamak ve yüksek hücre canlılığı korumak için mümkün olduğunca çabuk lenfositlerin tek hücre süspansiyonları üretmek için önemlidir. <ol><li> Servikal dislokasyon veya CO 2 boğulma tarafından deneysel fare Euthanize. Not: itibaren Bu adımdan tüm deneysel işlemler aseptik olarak yapılmalıdır. </li><li> Herhangi bir kesiler yapmadan önce% 70 etanol içine tüm fare batırın. Dalak ve lenf düğümleri aseptik 8 çıkarın ve ya (aşama 1.2.3 den) BSS / FBS (% 2 FBS ile RPMI) 5 ml buz gibi soğuk RPMI / FBS ihtiva eden ayrı ayrı 15 ml tüpler yerleştirin. Not: BSS etkin RBC parçalama engellediğinden, dalak hücre süspansiyonu hazırlamak için RPMI kullanmak afte BSS geçmekr RBC parçalama. </li><li> 2 ml buz gibi soğuk RPMI / FBS BSS / FBS içeren bir petri steril 100 um hücre filtresi örgü iki adet arasında organ (lar) yer, dalak ve lenf düğümleri, tek bir hücre süspansiyonu üretmek için. Çok ince parçalar halinde yırtılmış kadar 1 ml şırınga pistonu kullanarak, organı (ler) ezin. </li><li> 15 ml tüp hücre süspansiyonu aktarın ve hücre süzgecinden buz soğukluğunda olarak RPMI / FBS BSS / FBS ile örgü yıkayın. Kalan hücre süspansiyonu toplayın aynı 15 ml tüp ekleyin ve 4 ° C&#39;de 5 dakika boyunca 453 xg&#39;de aşağı doğru döndürün. süpernatantı. NOT: Sonraki adımda RBC parçalama tamponu veya orta eklemeden önce parmaklarıyla tüp hafifçe vurarak pelet hücreleri askıya Re. </li><li> Hücre süspansiyonu santrifüje esnasında oda sıcaklığında (RT) RBC liziz tamponu (adım 1.3 bkz.). Süpernatant hücrelerin peletleme ve çıkardıktan sonra, her 10 8 hücreleri için 1 ml RBC parçalama tamponu ile hücrelerin yeniden askıya. incukesmek 3-4 dakika boyunca oda sıcaklığında lizis reaksiyonu. </li><li> 14 ml buz gibi soğuk BSS / FBS ile RBC lizis durdurun ve 4 ° C&#39;de 5 dakika boyunca 453 xg&#39;de aşağı doğru döndürün. </li></ol> B ve T hücrelerinin 3. saflaştırılması <ol><li> B hücrelerinin saflaştırılması <ol><li> Bir hemasitometre kullanarak hücreleri saymak. 300 ul BSS / FBS kadar 10 8 dalak hücreleri yeniden askıya ve 50 ul anti-CD43 manyetik mikroboncukları 9,10 ekleyin. , Ölü hücreleri çıkarmak 30 ul Annexin V manyetik boncuklar ekleyin. 30 dakika boyunca 4 ° C&#39;de buzdolabında hücre süspansiyonu inkübe edin. </li></ol></li><li> T hücreleri ile saflaştırılması <ol><li> Bir hemasitometre kullanarak hücreleri saymak. Olmayan T hücre tükenmesi antikor kokteyli 10 8 hücreleri (CD19, B220, Gr-1 karşı biotinlenmiş antikorlar kadar yeniden askıya TCR-γδ, CD49b, CD11c, CD11b, Ter119 ve CD4 veya CD8 hedef hücre popülasyonuna bağlı olarak / FBS 4,5 200 ul BSS 200:) arındırılmalıdır, 1 seyreltilmiş. Bir hücre süspansiyonu inkübe15 dakika boyunca 4 ° C&#39;de bir buzdolabı. </li><li> İnkübasyondan sonra, hücreler yıkama ve 4 ° C&#39;de 5 dakika boyunca 453 x g&#39;de döndürülür ve 10 mi BSS / FBS ekleyin. Süpernatantı ve 30 ul streptavidin mikro boncuklar ve 15 ul Annexin V manyetik boncuklar ile 165 ul BSS / FBS hücreleri yeniden askıya. 30 dakika boyunca 4 ° C&#39;de buzdolabında hücre süspansiyonu inkübe edin. NOT: bile manyetik mikro-ile etiketleme sağlamak 15 dakika boyunca mikro-hücreleri inkübe sonra 15 ml tüp dokunarak yavaşça hücre süspansiyonu karıştırın ve adım 3.1.1 sırasında başka bir 15 dakika inkübe etmek. ya da 3.2.2. </li></ol></li><li> Hücre Arıtma için Ayırma Sütunu hazırlanması <ol><li> Hücrelerin microbead etiketleme sırasında kullanılmayan bir ayırma kolonu hazırlayın (adım 3.1.1. ya da 3.2.2.). RT&#39;ye (FBS olmadan) Pre-sıcak BSS ve yıkama ve aseptik sütun dengelenmeye 2 ml kullanın. BSS ile dengelendikten sonra, yıkanmış, kolon kurumasına izin verilmemelidir. NOT: LS c kullanınolumn yerine tavsiye edilen LD sütun nedeniyle yeniden kullanılabilirlik (adım 3.4.). </li><li> Manyetik boncuklar ile etiketleme işleminden sonra, hücreler yıkama ve 4 ° C&#39;de 5 dakika boyunca 453 x g&#39;de döndürülür ve 14 ml BSS / FBS ekleyin. Süpernatantı ve 1-3 ml RT BSS hücreleri yeniden askıya. </li><li> saflaştırma işlemi sırasında akış oranını azaltmak için sütunun ucuna steril 21 G iğne takın. dengelenmiş kolona hücre süspansiyonu yükleyin ve saflaştırılmış hedef hücreleri ihtiva eden akışı toplamak. NOT: sütuna yükleme sırasında içine kabarcıkları tanıtan kaçının. </li><li> 1 mi BSS / FBS ile bir kez kolon yıkaması ve saflaştırılmış hedef hücreleri ihtiva eden akışı toplamak. bir kez daha içinden akışı ile sütun yeniden yükleyin. Aynı 15 ml tüp ikinci yüklemeden sonra akışı toplayın. </li><li> 1 ml BSS / FBS ile sütun 3 kere yıkayın ve saflaştırılmış hedef hücreleri ihtiva eden akışı toplamak. Bundan sonra, th 5 ml BSS / FBS ekleyinBir dalgıç ile e kolon ve yeni bir 15 ml tüp içine sütun dışında manyetik etiketli hücreleri yıkayın. </li><li> Saflaştırılmış B veya T hücrelerinin 4,5 antijenlerini yüzey bağlanan antikorlar kullanılarak akış sitometrisi ile toplanan hücreler saflığını kontrol edin. </li></ol></li><li> Yeniden kullanılması ayırma kolonunun NOT: LS sütun arıtma verimini etkilemeden 4 kata kadar tekrar edilebilir. <ol><li> 5 ml fosfat tamponlu tuz (PBS) ve pistonu kullanan üstten damıtılmış su, 5 ml 3 kez sütun 3 kez yıkayın. </li><li> 5 ml% 70 etanol ile sütun yıkayın ve yoğun sütunda pas oluşumunu önlemek için bir hava musluğunu kullanarak sütun kurutun. </li><li> ayn bir saflaştırma deneyi için el LS kolonu hazırlamak için, şırınga adaptörü kullanarak 5 mi,% 70 etanol ile aşağıdan yukarıya doğru sütun yıkayın. Daha sonra, sütun üst bir kez 5 ml PBS ve ardından, 5 ml steril PBS ile iki kez aşağıdan yukarıya doğru sütun yıkayın. 2 ekleml RT BSS sütun dengelenmesi ve ardından etiketli hücreleri ile kolon yükleme devam etmek. </li></ol></li></ol> 4. KAKE Etiketleme ve Uyarım Not: Saflaştırılmış hücreleri, in vitro çeşitli ve in vivo fonksiyonel deneylerde tabi tutulabilmektedir. Burada, APC &#39;5 T hücresi uyarılması yeteneğini belirlemek için, saflaştırılmış T hücreleri kullanır. <ol><li> 37 ° C öncesinde CFSE yüklenmesi Pre-sıcak etiketleme çözümü (PBS içinde% 0.1 FBS). Not: PBS içinde FBS düşük bir yüzde kullanarak CFSE yükleme esnasında hücre ölümünü azaltır ve santrifüj sırasında hücre kaybını en aza indirir. Ancak, çok fazla FBS KAKE yükleme engelleyebilir. </li><li> Etiketleme çözeltisi ile iki kez saflaştırıldı hücreler yıkanır, daha sonra 15 ml&#39;lik bir tüp içinde önceden ısıtılmış markalama çözeltisi içinde 2 x 10 7 hücre / ml&#39;de yeniden süspanse edilir. </li><li> 10 uM KAKE çözeltisi hazırlayın (1: 500 seyreltme 5 mM KAKE stok solüsyon) önceden ısıtılmış etiketleme çözeltisi içinde. KAKE çözümü gerekirtaze optimum etiketleme elde etmek için her zaman hazır olun. </li><li> CFSE ile hücrelerin yüklemek için, 37 ° C &#39;de 10 dakika süre ile karanlıkta, 15 ml&#39;lik bir tüp içinde 1 kısım 10 uM CFSE çözeltisine 1 kısım hücre süspansiyonu ekleyin ve inkübe edilir. 5 uM CFSE bir son konsantrasyon elde 1 x 10 7 hücre / ml etiketlemek için kullanılır. </li><li> CFSE yükleme esnasında hücre, homojen bir karışım sağlamak için tüpü her 2 dakikada ters çevirin. </li><li> , Reaksiyonu durdurmak buz soğukluğunda tam RPMI ortamında bir çok sayıda ekleme ve 4 ° C&#39;de 5 dakika boyunca 453 x g&#39;de döndürülür ve. Başarılı KAKE yükleme üzerine, hücre pelet sarımsı görünecektir. </li><li> Yıkama CFSE buz soğukluğundaki tam RPMI ortamı ile hücreler bir kez daha yüklenir ve in vitro kültür için ya da in vivo uyarılması kullanılmadan önce, 4 ° C&#39;de 5 dakika boyunca 453 x g&#39;de aşağı doğru döndürün. </li></ol> 5. İn vitro uyarma <ol><li> kullanımdan hemen önce uyaranların 2x stok solüsyonu (2x uyaranlara stok çözelti) hazırlayın 10 ve böylece2x uyaranlara stok çözeltisi 0 ul, 96 oyuklu bir plaka içerisinde, oyuk başına 200 ul nihai bir hacme 100 ul hücre ilave edilebilir. </li><li> (B hücreleri ve T hücreleri için, CD3 ve CD28 için IgM veya CD40) uyaranlara ile kaplanmış bir tabak gerekli ise, bir gece boyunca 4 ° C&#39;de PBS içinde uyaranlara ve ön-kaplama kültürü plakası seyreltin. Seçenek olarak ise, kültür plakası Deney gününde 1 saat boyunca 37 ° C &#39;de kaplanabilir. PBS (plaka, herhangi bir zamanda kurumasına izin vermeyin) ile iki kez kaplanmış plaka yıkayın. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> B hücreleri için </td><td></td></tr><tr><td> uyaranlar </td><td> Nihai konsantrasyon </td></tr><tr><td> F (ab &#39;) 2 keçi anti-fare IgM </td><td> 0,6-2,4 ug / ml </td></tr><tr><td> Fare karşıtı CD40 mAb </td><td> 0.5-2 ug / ml </td></tr><tr><td> rekombinantfare IL-4 </td><td> 25 U / ml </td></tr><tr><td> lipopolisakkarit </td><td rowspan="2"> 0,1-10 ug / ml </td></tr><tr><td> E. (LPS) E. coli serotipi 055: B5 </td></tr><tr><td></td><td></td></tr><tr><td> T hücreleri için, </td><td></td></tr><tr><td> uyaranlar </td><td> Nihai konsantrasyon </td></tr><tr><td> Anti-CD3 (plaka kaplı) </td><td> 2-10 ug / ml </td></tr><tr><td> (50 ul / oyuk kaplama) </td><td></td></tr><tr><td> Anti-CD28 (plaka kaplı) </td><td> 2 ug / ml </td></tr><tr><td> Rekombinant IL-2 </td><td> 40 U / ml </td></tr><tr><td> PDBu (forbol ester) </td><td> 5-50 ng / ml </td></tr><tr><td> A23187 (Kalsiyum İyonofor) </td><td> 250 ng / ml </td></tr></tbody></table> Tablo 1: in vitro kültür içinde lenfositleri uyarmak için kullanılan uyarıcı konsantrasyonu.<ol start="3"><li> CFSE, etiketli B hücreleri için, 3 x 10 5 hücre ile üç nüsha tam RPMI ortamı ve kültürde 6 ila 10 x 3 hücre / ml yeniden askıya / kuyuda 72 saat için 96-gözlü düz tabanlı plakalar. </li><li> CFSE-etiketli T hücreleri için, 0.5-3 x 10 5 hücre ile üç nüsha tam RPMI ortamı ve kültür 0,5-3 x 10 6 hücre / ml&#39;de yeniden askıya / kuyuda 48 ya da 72 saat süre ile, 96-gözenekli yuvarlak tabanlı plakalar . </li></ol> 6. in vivo uyarılmasına <ol><li> In vivo uyarılmasına, adoptif transferi için fare başına 4 x 10 6 KAKE etiketli T hücreleri (intravenöz (iv) 200 ul PBS) her MHC-uyumlu alıcı fare içine. NOT: Bu protokol, KAKE etiketli T hücreleri adoptively ev gibi dalak ve lenf düğümleri olarak lenfoid organlara olacak bu hücrelerin olarak kuyruk ven ya da retro-orbital enjeksiyonu kullanılarak transfer edilebilir. </li><li> Bir gün sonra antijen ile alıcı fareler meydan. NOT:OVA-spesıfik T hücresi reseptörü (TCR) -transgenic T hücrelerinin adoptif alıcı farelere aktarıldı, bu örnekte, antijen olarak ovalbumin (OVA proteini, 50 ug / fare) kullanın. Her alıcı farenin 5 içine, deri altı enjeksiyon (si) üzerinden, steril PBS OVA proteini hazırlayın ve OVA protein / PBS veya PBS kontrol 100 ul enjekte edilir. </li><li> Hasat ve 3 gün OVA proteini veya PBS ile immünizasyondan sonra alıcı farelerin bir lenfoid organlara hücre süspansiyonları (lenf düğümleri ve dalak) oluşturur. proksimal lenf nodlarının mezenterik, popliteal, inguinal, lomber ve kuyruklu lenf düğümleri içeren aksiller, brakial ve yüzeysel servikal lenf düğümleri) ve distal lenf nodları (DLN) içerir (PLN), içine ayrı lenf nodları. Uygun FACS antikorları kullanarak leke hücreler, T hücre çoğalması kontrol etmek için. NOT: Bu KAKE hücre izleme deneyinde olmayan di için bir temel floresan kurmak PBS enjekte edilmiş kontrol farelerinden T hücrelerinin KAKE etiketliHücreleri sa¤layan. Çoğalan hücre bölünmeleri, antijen ile uyarılmış, KAKE etiketli hücreler floresan zirveleri 12,13 ölçülerek tarafından görüntülenmiştir. PBS kontrolü ile, çoğalan hücre bölünmeleri sayısı KAKE etiketli T hücreleri 12,13 belirlenebilir. </li><li> Örnekler arasında, hücre bölümleri veya tepe sayısını karşılaştırarak CFSE etiketli hücre proliferasyon verileri analiz (Şekil 1 ve 2). Not: Örneğin, CFSE etiketli, adoptif OVA antijen alıcı alıcı farelere aktarıldı OVA spesifik T hücreleri, PBS enjekte edilmiş kontrol farelerine kıyasla 5,12 aktif proliferasyonunu uğrayacaktır. Ayrıca, Hawkins ve arkadaşları 14 tarafından gösterildiği gibi KAKE hücre çoğalma verileri analiz etmenin birçok yolu vardır işaret etmek çekicidir. </li></ol>

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S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat. Protoc. 2 (9), 2049-2056 (2007).</li><li>Quah, B. J. C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> J. Immunol. Methods. 379 (1-2), 1-14 (2012).</li><li>Hawkins, E. D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, 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=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nat. Protoc. 2 (9), 2057-2067 (2007).</li><li>Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation:+Terminology+and+practical+considerations.">Cell separation: Terminology and practical considerations.</a> J. Tissue Eng. 4 (1), 1-14 (2013).</li></ol>

The authors have nothing to disclose.

Bu protokol, lenfoid organların lenfositlerin saflaştırılması için bir prosedürü göstermektedir. manyetik boncuk sıralama kullanılarak hücre arıtma canlı, yüksek düzeyde saflaştırılmış hedef hücreleri verimleri hızlı ve basit bir yöntemdir. Protokol çerçevesinde kritik adımlar Hücre canlılığı, ve hücre verimi In vitro olarak hematopoietik kökenli hücrelerin canlılığını m...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640 (without L-Glutamine)</td>
			<td>Gibco</td>
			<td>31870025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td></td>
			<td></td>
			<td>Heat inactivated&#160;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140114</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD43 magnetic microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-801</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Streptavidin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>Anti-Annexin V magnetic beads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-201</td>
			<td>Mix well prior use</td>
		</tr>
		<tr>
			<td>MACS LD&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>650180</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well F-bottom sterile culture plate</td>
			<td>Greiner Bio-one</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer mesh</td>
			<td></td>
			<td></td>
			<td>To sterilize using UV radiation prior use</td>
		</tr>
		<tr>
			<td>0.2 &#956;m&#160; sterile disposable filter units</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td>Can be substituted with any sterile filter device</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td>CTV for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Yellow</td>
			<td>Invitrogen</td>
			<td>C34567</td>
			<td>CTY for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>CellTrace Far Red</td>
			<td>Invitrogen</td>
			<td>C34564</td>
			<td>CTFR for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>Cell Proliferation Dye eFluor 670</td>
			<td>eBioscience</td>
			<td>65-0840</td>
			<td>CPD670 for short; alternative to CFSE</td>
		</tr>
		<tr>
			<td>PKH26</td>
			<td>Sigma Aldrich</td>
			<td>PKH26GL</td>
			<td>PKH26, alternative to CFSE</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck Millipore</td>
			<td>S7653</td>
			<td>Can use from other sources</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>Molecular Probes</td>
			<td>C-1157</td>
			<td>Reconstitute in DMSO</td>
		</tr>
		<tr>
			<td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td>
			<td>Sigma Aldrich</td>
			<td>P1269</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (Calcium ionophore)</td>
			<td>Sigma Aldrich</td>
			<td>C7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies and recombinant protein</td>
		</tr>
		<tr>
			<td>CD11b biotin (clone m1/70)</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD11c biotin (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Gr-1 biotin (clone RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>Ter119 biotin (clone Ter119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948; biotin (clone GL-3)</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD19 biotin (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>B220 biotin (clone RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD49b biotin (clone DX5)</td>
			<td>Biolegend</td>
			<td>108904</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD4 biotin (clone GK1.5)</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>CD8 biotin (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>T cell depletion cocktail</td>
		</tr>
		<tr>
			<td>F(ab&#8217;)2 goat anti-mouse IgM (plate coated)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>115-006-075</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD40 mAb (plate coated)</td>
			<td>Pharmingen&#160;</td>
			<td>553722</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-4</td>
			<td>ProSpec&#160;</td>
			<td>Cyt-282</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS from E. coli Serotype 055:B5</td>
			<td>Sigma Aldrich</td>
			<td>L-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3 (clone clone OKT3) (plate coated)</td>
			<td>eBioscience&#160;</td>
			<td>16-0037-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Anti-CD28 (clone clone 37.51 ) (plate coated)&#160;</td>
			<td>eBioscience&#160;</td>
			<td>16-0281-85</td>
			<td>50 &#181;l/well for coating (96-well)</td>
		</tr>
		<tr>
			<td>Recombinant IL-2</td>
			<td>ProSpec</td>
			<td>Cyt-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from chicken egg white, Ovalbumin</td>
			<td>Sigma Aldrich</td>
			<td>A7641</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and activation of murine lymphocytes

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

lenfositlerin manyetik hücre saflaştırma kullanıcılara zaman nispeten kısa bir miktarda bir hedef hücre popülasyonu arındırmak için izin verir. Bizim tükenmesi protokolü kullanarak, arıtıldıktan sonra 94.2% (için 72,8% (önceki arıtma) den (rekombinasyon aktive gen 1 (RAG-1) -açıklı farelerde OT-I) CD8 T hücrelerinin yüzdesini artırmak başardık; Şekil 1A) 4,5. Bu arıtılmış, lenfositler, daha sonra olan limfosit proliferasyon ve s...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

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

İzolasyon ve murin lenfositlerin aktivasyonu

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

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

20.9K Görüntüleme.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Video: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Murin Model B hücreleri CD40 aktivasyonu

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

İmmünoloji ve Enfeksiyon

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Lenfositler ve mürin Peyer en Yamalar itibaren Dendritik Hücreler İzolasyon Ve immün

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation 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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T Lenfositlerindeki Integrin Aktivasyonunun Çalışması için statik Adezyon Deneyi

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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

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

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

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

Isolation of Murine Lymph Node Stromal Cells

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

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

Kemirgen Lenf Düğümü Stromal Hücrelerinin İzolasyonu

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

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

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 ...

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.

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 ...