Bu çalışma (Kah için) Sağlık Hibe R01 AI088209 Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Biz de Minnesota Sitometrisi Kaynağın Üniversitesi teşekkür ederim.

Bu çalışmada, yetişkin (6-8 haftalık) dişi C57BL / 6 fareleri kullanılmıştır. Fareler, Ulusal Kanser Enstitüsü alınan ve spesifik patojensiz şartlar altında muhafaza edilmiştir. Minnesota Kurumsal Hayvan Bakım ve Kullanım Komitesi Üniversitesi (IACUC) bütün hayvan deneylerini onayladı. Araçların ve Tamponlar hazırlanması 1. <ol><li> Enzim çözeltisi hazırlayın, albüminsiz açısından zengin tamponlu (1X PBS: [Ca2 + / Mg2 +-free]% 0.5 sığır serumu (% 0.05 içeren RPMI1640 ortamı Liberase® TH ve DNase I 100 U / ml [w / v]) albümini ve 2 mM etilendiamin tetraasetik asit [EDTA]) FACS tampon (PBS 1X [Ca2 + / Mg2 +-free] ihtiva eden,% 1 fetal inek serumu [FBS] ve% 0.02 NaN3), tampon sıralama FACS (1X PBS [Ca2 + / Mg2 +-free]% 2 FBS) ve RP10 ortamında içeren (RPMI-1640 ortamı),% 10 FBS ile birlikte. </li><li> 10 kaydırma tabak hazırlayın. (0.01 mg / ml G 40 mi kaplama çözeltisi hazırlayınyulaf, 0.1 M NaHCO3 Tamponu içinde anti-fare IgG) ve her biri 100 x 15 mm levha, girdap içine kaplama solüsyonu ve 4 ml 4 ° CO / N inkübe edilir. Antikor çözüm dökün ve 2 kez 1x 5 ml PBS kullanılarak plaka durulama, durulama arasındaki şiddetle girdap. 4 ° C &#39;de hazırlanmış plakalar saklayın. </li></ol> Thymus Doku 2. Hasat <ol><li> Bir CO2 odası içinde fare öldürülür. , Bir diseksiyon mat sırtında hayvan koymak mat ayak pin ve% 70 etanol ile püskürtülerek kürk ıslak. </li><li> Düz kadar makas ile alt çene için üretral açıklıkta yukarıdaki başlayarak, deri yoluyla bir ensizyon olun ve her iki tarafta diz aşağı kesi uzatmak. Kesi &quot;Y&quot; bir baş aşağı gibi görünüyor. Tarafta gevşek deriyi geri çekin ve diseksiyon mat onu pin. </li><li> , Kılıç şeklinde diyaframı delinme klavikula hakkında, daha sonra kaburga ile yukarı kaldırın kadar her tarafta kaburga kestiforseps diseksiyon mat onu pin ve. Timus sadece kaburga altında ve kalp (Şekil 1) örten ince iki beyaz lob gibi görünüyor. Kes bağ dokusu hafifçe çekin ve kavisli dişli forseps ile timus lobları çifti kaldırmak, timus çevreleyen. 5 ml RPMI-1640 ihtiva eden bir 6 yuvalı plaka içine yerleştirin timus loblar. </li></ol> Timik Stromal Hücrelerinin 3. hazırlanması <ol><li> Çevredeki yağ ve bağ dokusu Trim ve 5 ml taze bir enzim solüsyonunun hazırlanması ihtiva eden 6 oyuklu plakanın yeni bir oyuğuna timus lobları aktarın. Ince bir makas timus loblarda küçük kesikler yapmak ve 20 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Doku ayrışması için 5 ml pipet kullanarak 3 kez - yavaşça timus doku yukarı ve aşağı 2 sindirerek pipetle. 10 ml enzimleri etkisiz hale getirmek için soğuk albümin açısından zengin tampon içeren 50 ml&#39;lik bir tüp içinde süpernatan fraksiyonu toplamak. Buz üzerinde toplama tüpünü tutun. 2.5 Eklemi enzim kalan dokuya çözeltisi ve 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Agrega kırmak için 3 ml şırınga ve 18 G iğne ile hafif mekanik ajitasyon gerçekleştirin. Toplama tüpüne Havuz süpernatant. Ve geri kalan dokulardan 2.5 mi enzim çözeltisi eklenir ve 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> 3 ml şırınga ve 25 G iğne ile nazik mekanik ajitasyon tekrarlayın ve plaka ek 5 kuluçkaya - 10 dk. </li><li> Tam bir sindirimden sonra, toplama tüpünün içine süpernatant bir araya getirirler. Santrifüj 400 xg, 8 dk hücreleri toplamak için 4 ° C&#39;de toplama tüpü. Aspire sıvı ve 10 ml albümin zengin tamponu içinde topak hücrelerin süspansiyonu ve 100 mm-örgü genişliği ile örnek filtre edin. Hemasitometre kullanarak hücreleri saymak. Sonra akış cytometic analizi (Bölüm 4) veya Tecs zenginleşme (Bölüm 5) gerçekleştirin. </li></ol> Sitometrisi ile Tecs 4. Kimlik </p&gt; <ol><li> 96 havuzlu yuvarlak dipli deney plakasında boyanması için FACS tampon maddesi içinde 100 ul başına 4 x 10 6 hücre arasında nihai bir konsantrasyona kadar hücrelere hazırlamak ve buz üzerinde plaka tutun. Hücrelere (10 ug FACS tamponu / ml anti-CD16 / CD32 antikoru) 20 ul Fc blok çözeltisi eklenir ve buz üzerinde 10 dakika için kuluçkalayın. 400 x g&#39;de 3 dakika boyunca 4 ° C &#39;de plaka Spin. Bir lavabo içine plaka yüzeyini hafifçe vurarak kuyulardan sıvı atın. </li><li> Tanelenmiş hücreler 100 ul antikor kokteyli içinde (anti-CD45 -EpCAM, MHC sınıf II, -Ly51 antikorlar ve farklı üreticiler florokromlar ile işaretlenmiş UEA-1 lektin önerilen ya da FACS tampon her bir antikorun uygun konsantrasyonlarda test Askıya ) ve karanlıkta 20 dakika süreyle buz üzerinde kuluçkalayın. Telafisi için, her bir fluorokrom ile 1 x 10 6 hücre leke. </li><li> Her bir oyuğa FACS tampon 100 ul ve 400 x g&#39;de 3 dakika boyunca 4 ° C &#39;de plaka dönerler. Yıkama adımı birini tekrarlayınsaat ve daha sonra 100 ul FACS tampon maddesi içinde tekrar süspansiyon hücreleri. 300 ul FACS tampon maddesi ile doldurulmuş bir 12 x 15 mm yuvarlak dipli bir FACS tüpü hücreleri aktarın. Sitometresiyle akış örnekleri Edinme ve FACS analiz yazılımı kullanarak verileri analiz. TEC yolluk strateji Şekil 2&#39;de gösterilmiştir. </li></ol> Panning ile Tecs 5. zenginleştirilmesi <ol><li> 400 x g&#39;de 5 dakika boyunca 4 ° C &#39;de (Bölüm 3&#39;te hazırlandı) başlanarak ve timus hücreleri dönerler. Aspire sıvı ve bir 15 ml ve 50 ml konik bir tüp içinde ml başına 2 x 10 8 hücre konsantrasyonunda FACS tamponu içerisinde sıralama topak hücrelerin süspansiyonu. Hücre süspansiyonu, 2.5 mikrogram / ml lik bir nihai konsantrasyonda anti-CD90.2 antikoru (klon 30-H12) (ya da anti-CD45) ilave edilir ve 30 dakika boyunca 4 ° C&#39;de dönen bir miksere yavaşça kuluçkalayın. Kuluçkalamanın ardından, 5 ile yıkayın - RP10 ortamında 10x hacmi ve 400 x g&#39;de 5 dakika boyunca 4 ° C&#39;de santrifüj tüpü. </li><li> Aspire sıvı ve pelet hücreleri tekrar süspansiyon, Ml başına 1 x 10 7 konsantrasyonunda RP10 ortamında. Her bir kaplanmış kaydırma (Kademe 1.3 hazırlandı) levha, girdap 5 mi hücre süspansiyonu ekleyin ve oda sıcaklığında 30 dakika inkübe edilir. Şiddetle girdap; konik bir tüp içine süpernatantlar aktarın. Plakalı RP10 ortamı kullanılarak iki kez durulayın ve toplama tüpünün içine Süpernatantlar havuzu. </li><li> 400 x g&#39;de 5 dakika boyunca 4 ° C&#39;de santrifüj toplama tüpü. Aspire sıvı ve 5 ml RP10 ortamında yeniden süspanse pelet hücreleri. Yeni bir kaydırma plakasına hücre süspansiyonu ekleyin girdap ve oda sıcaklığında 30 dakika inkübe edilir. Hücreler plaka yıkayın ve konik bir tüp içine havuzu toplayın. Tecs zenginleştirilmiş sonra, tampon sıralama 5 mi FACS hücreler 400 x g&#39;de 5 dakika boyunca 4 ° C&#39;de hücreleri durması ve askıya. </li></ol> Hücre sıralayarak Tecs 6. saflaştırılması <ol><li> Hemasitometre ile hücre sayımı ve FACS tamponu içerisinde 4 x 10 7 hücre bir konsantrasyonda bir hücre süspansiyonu hazırlanır. Antikorları des EkleHücre çözeltisi içine, Bölüm 4.4 &#39;de tanımlanmış ve 30 dakika boyunca 4 ° C&#39;de dönen bir miksere yavaşça kuluçkalayın. Boyamayı takiben, yıkama ve FACS tampon ml&#39;si başına 10 x 10 6 hücre bir konsantrasyona hücrelerin süspansiyonu. </li><li> Sıralama, 100 uM alt-gruplar meme boyunca TEC fluoresan ile etkin hale getirilmiş hücre sıralaması kullanılmıştır. Kriteri hücre, RPMI-1640 içinde (hac / hac) FBS) içeren% 30 toplanır. Hücre sıralama 400 x g&#39;de 5 dakika boyunca 4 ° C&#39;de santrifüj yapılır ve hücreler alt analizi için hazırlamak sonra. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoepithelial+interactions+in+the+mouse+thymus:+phenotypic+and+kinetic+studies+on+thymic+nurse+cells.">Lymphoepithelial interactions in the mouse thymus: phenotypic and kinetic studies on thymic nurse cells.</a> J Immuno. 128, 2287-2294 (1982).</li><li>Nakagawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+nurse+cells+provide+microenvironment+for+secondary+T+cell+receptor+alpha+rearrangement+in+cortical+thymocytes.">Thymic nurse cells provide microenvironment for secondary T cell receptor alpha rearrangement in cortical thymocytes.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 20572-20577 (2012).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J. Immunol. Method. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of immunological method. 385, 23-34 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Protokolde, kritik adımlar timus stromal hücrelerinin (Bölüm 3) hazırlama ve Tecs zenginleştirme (Bölüm 4) vardır. Kuvvetle taze enzim solüsyonu her zaman hazırlanır ve doku kısa sürede tedavi tavsiye edilir. Toplanmış Thymi için, enzim çözeltisinin hacmini optimize Thymi sayısına bağlı olarak gereklidir. Artık madde, herhangi bir doku, işlemden sonra kalan, daha fazla enzim çözeltisi ve inkübasyon süresi uzantısı ekleyerek hücre verimini artırabilir. Tecs zenginleştirilmesi, süpernata...

Erken T hücresi gelişiminde, kemik iliği hematopoetik kök hücre türevli multipotent progenitorlar timus korteks için işe, T kökene bağlı geçmesi ve T hücresi ön-1 olur. Son derece değişken T hücre reseptörleri 1 progenitörlerin büyük bir havuz oluşturan, kortekste T hücresi öncü CD4 ve CD8 çift negatif (DN), timositler genişletmek ve olgun CD4 ve CD8 çift pozitif (DP), timositler içinde farklılık gösterir. Sadece DP hücrelerin seçilmiş bir MHC ile sınırlandırılmış bir alt kümesi, CD4 veya CD8 tek bir pozitif (SP), timositler hale timus medullaya geçer ve fonksiyonel olarak yetkin olgun T hücrelerine gibi pozitif seleksiyona 2 adlandırılan bir olay ayırt edecektir 6. Buna karşılık, oto-reaktif timositlerin klonları negatif seçim geçmesi ve öz-hoşgörü için düzenleyici T hücreleri dönüştürülür, ya da henüz net değil amaçlarla intraepitelyal lenfositlerin yönlendirilir, apoptoz yoluyla uzaklaştırılır 3,710. Timus, timik stromal hücreler bu çeşitli T hücre gelişimi kaderi 5,11,12 için sinyalleri veren eşsiz bir mikroortam oluştururlar. Kortikal Tecs (cTECs) ve kemik iliği Tecs (mTECs), dendritik hücreler, makrofajlar, fibroblastlar, endotel hücreleri, nöral tepe türetilmiş perisitlerin ve mezenkimal hücreler de dahil olmak üzere 13-15 - Timus, stromal hücreleri, timik epitelial hücreler üzerinde (Tecs) oluşmaktadır. Bunlar arasında, Tecs T hücresi gelişme 1,2,16,17 çeşitli aşamalarında çok önemlidir. Ancak, Tecs izole etmek için sağlam bir yol olmaması işlevleri 16 kapsamlı bir anlayış engelliyordu. Özellikle, kortekste ata hücreleri çevreleyen, üç boyutlu bir ağ oluşturmak cTECs, henüz net değildir nedenlerle pozitif seçim 13,18,19 için gereklidir. Daha önceki çalışmalar çoğunlukla morfolojik ve histolojik araçları 13 güvenerek, Tecs heterojen ve rolü gibi ipuçları sağlanan </sus&gt;. Son zamanlarda TEC alt kümelerinin eşsiz rolleri fare modellerinde 12,20 genetik yaklaşımlar ele alındı. Tecs izole etmek sağlam ve tekrarlanabilir yoldur cTECs pozitif seçim nasıl desteklediğini TEC alt kümeleri, TEC fonksiyonların nicel ve nitel değerlendirilmesi ve mekanizmaların aydınlatılmasını tarafsız karakterizasyonu elde esastır. Nedeniyle onlar bozulmamış organ formu timus Tecs azlığı ve sıkı etkileşimler, Tecs izolasyonu zorlu olmuştur. Burada açıklanan protokol, önceki keşifler, mevcut reaktif, teknikleri, ve timus yapısı ve stromal kompozisyon bilgisine dayanmaktadır. Yaklaşık iki yıl önce, birkaç prosedür tripsin, Kollajenazının ve Dispase dahil olmak üzere farklı enzimler sindirimi sırasında kullanılan edildiği timus dokuları 21-27, parçalamak bildirildi. Gray ve ark. kendi Muhakemelerine 28 bu enzimler göre ve geliştirilmiş bir meth Başkaoldu enzim kokteyller 29 bir çoklu-aşamalı sindirim od yaygın 20,30 kullanılmaktadır. Ancak, bu yöntem uzun bir işlem süresi ve hatta aynı sıçangil kohort 29,30 hücre sayıları ve Tecs oranlar son değişken karmaşık sindirim adımlar ve sonuçları içerir. Birkaç yıl önce, Liberase® araştırma sınıfı enzim, son derece saf hale getirilir ve kolajenaz nötr proteaz timus doku ayrışma 30 kullanılmaya başlanmıştır içeren. Burada, fare timusundan dokulardan uygulanabilir Tecs yüksek numarasını verir iyileştirilmiş mekanik ayırma prosedürleri ile sindirim Liberase® tabanlı bir protokol tanımlıyoruz. . Ayrışmış stromal hücreler zenginleştirmek için, daha önceki çalışmalar yoğunluk geçişlerini veya manyetik boncuk ayırma 21,29 ya kullandık. Ancak yöntem hem timik stromal hücreleri, cTECs 29 özellikle nüfusun belli popülasyonlarının kaybetti ciddi neden olur. Sitotoksik eliminasyon ve kaydırma teknikleri <s&gt; 31,32 kadar yaygın immünolojik alanında 12,31 tüketilmesi veya lenfositlerin artan ayrılması için kullanılmıştır. Bu tekniklerin karşılaştırılması sonra, biz Tecs zenginleştirilmesi için geçerli kaydırma protokolünü kurulmuştur. Zenginleştirme işlemi sırasında nazik durum daha az hücre ölümü ve tarafsız ve artan TEC kurtarma yol açar. Bölüm 3&#39;te tarif edildiği gibi izole edilmiş bir hücre süspansiyonu, timus, doğrudan alt-TEC ve dendritik hücrelerin tanımlanması ve karakterizasyonu için akış sitometrik analizi, uygulanabilir. Bölüm 4 sitometresini multiparametre akışını kullanarak TEC alt kümelerini tanımlamak için basit ve kullanışlı bir yol. Saflaştırılmış cTECs veya mTECs, TEC zenginleştirme ve ayıklama prosedürleri hücresi elde etmeye deneyler için Bölüm 5 ve 6&#39;da bulunabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

Timus T-lenfosit gelişimi için hayati bir organdır. T hücre bağlılık, pozitif ve negatif seçim seçimi: timik stromal hücrelerin, timik epitel hücreleri (Tecs) T hücre gelişiminin çeşitli aşamalarında özellikle önemlidir. Bununla birlikte, timus Tecs işlevi tam olarak anlaşılamamıştır. Makalede, mekanik parçalama ve enzimatik sindirme bir kombinasyonu kullanılarak taze fare timusundan TEC alt kümelerini izole etmek için bir yöntem sağlar. Yöntem, timus hücreleri ve antikor fragmanlarının verimli hücre-hücre ve hücre-hücre dışı matris bağlantıları serbest kalmasına ve tek bir hücre süspansiyonu meydana getirmek üzere sağlar. Izole edilmiş hücreler kullanılarak, çok parametreli akış sitometri Tecs ve dendritik hücrelerin belirlenmesi ve karakterize edilmesi uygulanabilir. Tecs timus nadir hücre topluluğu olduğundan, daha da zenginleştirmek ve timositleri, timus içinde en bol hücre tipi tüketerek Tecs arındırmak için etkili bir yol tarif eder. Follohücre canlılığı kaybının Tecs saflaştırılması sırasında minimize edilebilir, böylece kanat zenginleştirme, hücre tasnifi, ayırma süresi azaltılabilir. Saflaştırılmış hücreleri Real Time PCR, Western blot ve gen ekspresyonu profillemesi gibi çeşitli alt analizler için uygundur. Protokol TEC fonksiyonun araştırma ve hem de in vitro T hücresi sulandırma gelişimini teşvik edecektir.

Elde edilen hücre süspansiyonu timositler, hematopoietik kaynaklı stromal hücreler ve hematopoietik olmayan stromal hücrelerin oluşuyordu Bölüm 3&#39;te belirtildiği gibi, bu protokol kullanılarak, bir timüs bir organ, bir yetişkin fare (Bölüm 2) ve bir timüs hücre süspansiyonu uzaklaştırılmıştır hazırlandı. CD45 hematopoietik bir Pan-işaretleyici olarak timositlerde ve örneğin makrofajlar ve dendritik hücreler gibi hematopoietik hem de stromal hücrelerin üzerinde ifade edilir. Bunun aksin...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Burada izolasyonu, tanımlanması ve fare timik epitelial hücreler üzerinde (Tecs) saflaştırılması için etkili bir yöntem tarif eder. Bu protokol, normal T-hücresi gelişimi, timus disfonksiyon ve T hücresi sulandırılacak timus fonksiyonunun çalışmaları için kullanılabilir.

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-identification-purification-murine-thymic-epithelial

Research

JoVE Journal

Immunology and Infection

31.7K Görüntüleme.  University of Minnesota.  Burada izolasyonu, tanımlanması ve fare timik epitelial hücreler üzerinde (Tecs) saflaştırılması için etkili bir yöntem tarif eder. Bu protokol, normal T-hücresi gelişimi, timus disfonksiyon ve T hücresi sulandırılacak timus fonksiyonunun çalışmaları için kullanılabilir. 

Video: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Fare Uygulama M hücreleri tarafından Bakteriyel Alımı değerlendirin Peyer Patch Bağırsak Loop Testi bağlandı

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

İmmünoloji ve Enfeksiyon

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Fonksiyonel ve Moleküler Çalışmalar Birincil murin Tip II alveol epitel hücrelerinde akım sitometri İzolasyonu

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

İnsan Thymus ile myeloid dendritik hücreleri ve epitel hücreleri İzolasyonu

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

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 ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

EAK 16-II ile FACS Arıtılmış Timik Epitel Hücreleri 3-D Toplama Teşvik / EAKIIH6 Kendi toplanan Hidrojel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Teşhis edilmesi için kullanılan ilişkili Viral ve hücresel proteinler Viral DNA'ın arıtma

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.