Biz Dr teşekkür ederiz. Belirteçlerin temin Markus Manz ve Irving Weissman. Biz de sırasıyla Tıp ve NTU hastanenin Ulusal Tayvan Üniversitesi Koleji&#39;nde Akış Sitometrik Analizi ve Birinci Hücre Sıralama Çekirdek Tesisi ve İkinci Çekirdek Laboratuvarı tarafından sağlanan hizmeti kabul. Bu çalışma Bilim ve Teknoloji, Tayvan Bakanlığı tarafından desteklenen (EN 102-2320-B-002-030-My3) ve Ulusal Sağlık Araştırma Enstitüleri, Tayvan (IHUK-EX102-10256SI ve IHUK-EX103-10256SI).

C57BL / 6 vahşi tip fare, National Laboratory Animal Center (NLAC), Tayvan &#39;den satın alınmıştır. Tüm fareler yetiştirilir ve belirli bir patojen içermeyen koşullar altında tutuldu. Protokoller ve hayvan kullanımı prosedürleri gözden ve Tıp Ulusal Tayvan Üniversitesi Koleji Kurumsal Hayvan Bakım ve Kullanım Kurulu (İzni No: 20120075) tarafından onaylanmıştır. Ayrıca, araştırmacılar deneyler yaparken hayvanlarda ağrı, acı veya sıkıntı potansiyelini azaltmak için her türlü çabayı. Eldiven yukarıda açıklandığı Tüm işlemler oda sıcaklığında gerçekleştirilmiştir. AC-6 Besleyici hücreler hazırlanması 1. Not: AC-6.21 (AC-6 kısaca) (I. Weissman, Stanford Üniversitesi tarafından temin edilmiştir) stromal hücre hattı 18 RPMI içinde muhafaza edilmelidir 15% ısı ile inaktive edilmiş fetal büyükbaş hayvan serumu (FBS) ile takviye edilmiştir. Besleyici hücreler olarak hizmet etmek, AC-6 hücreleri-γ ışınlanmış 3000 rad (30Gy) onların çoğalmasını önlemek için ko-kültür bir gün önce ile vardır. Not inciOnların geçit sayısı 20 üzerinde ise ac-6 hücre bakım sırasında kalabalık bırakılırsa kendi farklılaşma destekleyen yeteneğini kaybetmek eğilimindedir ya. <ol><li> Yıkama AC-6 hücreleri, bir sefer 1 mL Dulbecco fosfat tamponlu tuz (DPBS) ile aspirasyon ile DPBS çıkarın. AC-6 37 o C&#39;de 3-5 dakika boyunca 0.8 ml tripsin çözeltisi (% 0.05 tripsin ve DPBS içinde 0.5 mM EDTA), sonra% 5 CO2 ve hücreler kültür ortamı 10 hacim için ile seyreltilmesi, reaksiyonu durdurmak Treat tek bir hücre süspansiyonu yapmak. </li><li> 5.9x10 tohum AC-6 hücre 4 hücre / kuyu içine 12-kuyu plakaları ve% 5, 37 o C&#39;de inkübe CO 2 O / N. Not: AC-6 hücrelerinin yoğunluğu cdc nesil lehine olacak bir alt yoğunluğu gibi çok önemlidir. 5.9x10 yoğunluğunda Tohum de AC-6 CLPs gelen pDC&#39;lerde türetilmesi için optimal olan bir sonraki gün, birleşik duruma ulaştılar sağlayacak / 4 hücreleri. </li><li> Γ-ışınlama ile 3000 rad (30 Gy) AC-6 hücreleri ışın tedavisi. Not: Ac-6 ışın tedavisi için kullanılan doz, çoğalan hücreler önlemek ve henüz CLPs DC&#39;lerin farklılaşması için gereken sitokinleri ve hücre-hücre teması sağlamak için yeterince uzun bir canlı tutmak için optimize edilmiştir. </li><li> Kültür ortamı basınçla (% 10 ısı ile inaktive edilmiş FBS, 50 ug / ml gentamisin ve 50 uM β-mersaptoetanol ile RPMI) tam RPMI 1 ml ile değiştirin. </li></ol> Fare 2. İzole BM Hücreleri <ol><li> CO 2 boğulma ve servikal dislokasyon ile fareler Kurban. Bir diseksiyon tepsiye yerleştirin fareler ve% 70 etanol ile sterilize edin. Orta karın bir kesi yapın ve alt ekstremite cilt kapsayan dahil farenin distalinde cilt kaldırın. </li><li> Yarı steril kaput fareler parçalara ayır. Femur ve fibula bırakın, diz ekleminin altında yukarıda femur ve fibula klibi. Makas kullanarak kemiklerden kasları ayırın ve RPMI tamamlamak ml 5 içeren 6 cm&#39;lik petri kemikleri yerleştirin. </li><li> Bir steril kültür kaputu taşıyın. Tam RPMI ile 27G iğnesi bağlı bir 3 ml şırınga doldurun. Makas kullanarak kemiklerin iki ucunu kesti ve yavaşça her iki ucundan bir kez tam RPMI enjekte edilerek kemiklerin dışarı iliği temizlemesini 27G iğne takın. </li><li> Hücreler yukarı ve aşağı 3-5 kez iğne olmadan şırınga kullanılarak kültür çanak nazik pipetleme tek bir hücre süspansiyonu hazırlayın. </li><li> 500 x g&#39;de 5 dakika boyunca santrifüj hücreleri ve süpernatantı süzün. </li><li> 1 ml tampon maddesi ACK (150 mM NH4CI, 10 mM KHCO 3, 0.1 mM EDTA) 1 dakika için inkübe edilmesi ve tam RPMI 10 hacim ile inceltilerek reaksiyon durdurularak ile lizleyin kırmızı kan hücreleri. Hücreler yerçekimi tarafından ölü hücre kümeleri ve doku artıkları aşağı pelet için 5 dakika bekletin. Not: Bu nedeni canlı hücrelerin yerleşme hücre kaybına neden olacak gibi en fazla 5 dk durmayın. </li><li> Yeni bir tüp bırakarak enkaz yavaş yavaş decant hücre içeren süpernatantOrijinal tüp arkasında. </li><li> Daha sonra, pelet hücreleri çıkarmak ve süpernatant atmak için 500 xg&#39;de 5 dakika santrifüj. </li><li> Toplam dengeleme hücrelerin tekrar FACS tampon maddesi (1x PBS +% 2 FBS + 1 mM EDTA) ul 100 (tipik olarak 4-6 x 10 7 BM hücreleri bir fareden elde edilir). </li></ol> 3. FACS Analizi ve CLPs sıralanması <ol><li> Fc reseptörleri bloke etmek için 1-2 dakika için, anti-CD16 / 32 (hibridom üstte yüzen klonu 2.4G2, 50 ul / tepkime veya 1-2 ug / reaksiyonu) FACS tampon maddesi içinde hücrelere (adım 2.9) ekleyin. Not: Anti-CD16 / 32, aynı zamanda granülositler, monositler ve B lenfositleri içeren Fc reseptörlerini eksprese eden hücrelere antikorların spesifik olmayan bağlanmasını önlemek üzere ilave edilir Fc blok olarak adlandırılır. </li><li> Aynı zamanda ortam ışığını kaçınarak, buz üzerinde 15 dakika süre ile (4x10 7 hücreleri için) Aşağıdaki fluoresan boya-etiketli antikorlar ile hücreleri boyayan. Antikorlar: PE-konjuge soy belirteçleri, anti-CD3 içeren (0.2 ug her biri) (17A2), anti-CD8 (53-6,7), anti-B220 (RA3-6B2), anti-CD 19 (eBio1D3), anti-CD11b (M1 / 70), anti-Gr-1 (RB6-8C5), anti- Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), ve anti-MHC-II (Nimr-4), 0,2 ug anti-c-Kit PerCP Cy5.5- (2B8), 1 ug anti-Sca-1-FITC (D7), 0.4 ug anti-M-CSFR-APC (AFS98) ve 0,2 ug anti-IL-7Rα-PE-Cy7 (A7R34). </li><li> 500 x g&#39;de 5 dakika boyunca 3 ml FACS tamponla hücreleri ve santrifüj yıkayın. </li><li> 300 ul FACS hücreler 40 mcM hücre süzgecinden tampon ve filtre hücreleri tekrar süspansiyon. </li><li> Kalan hücrelerin kurtarmak ve aynı hücre süzgeci kullanılarak tüp ayırma steril FACS nüfuz etmeye ek bir 100 ul FACS tamponu ile tüp yıkayın. </li><li> Sinyal tespiti için uygun filtre ve voltajlar kullanılarak boyanması hemen sonra akım sitometri analizi yapın. PE- ve PE-Cy7-labelded antikorları ve 633 nm las algılamak için 561 nm lazer, FITC-, PerCP-Cy5.5 etiketli antikorları tespit etmek için 488 nm lazer kullanıner APC-etiketli antikor tespit etmek için. </li><li> C-kit int Sca-1 int M-CSFR - - IL-7Rα + (adım 3.2 lekeli gibi) hücre sıralayıcı kullanılarak sırala dışarı CLPs aşağıdaki belirteçler lin göre. Bir yastıkta tam RPMI 8 ml içeren 15 ml tüp sıralanır hücreleri toplayın. </li><li> Çalışmasının sonunda hücre sıralayıcı ile gösterildiği gibi kriteri hücrelerinin mutlak sayısı kaydedilir. Tipik haliyle, 5x10 4 CLPs bir fare BM&#39;lerine elde edilebilir. </li></ol> 4. Coculture CLPs ve AC-6 <ol><li> , 500 x g&#39;de 10 dakika boyunca aşama 3,7 kriteri hücreleri Santrifüj süpernatantı çıkarın ve 5x10 4 hücre / ml civarında bir hücre yoğunluğuna elde etmek için yeterli tam RPMI hücre pelletini. Tohum 500 hücre / çukur olarak aşama 1.4&#39;te hazırlanan besleyici hücreleri ihtiva eden 12 oyuklu plaka. </li><li> Her bir oyuğa 100 ng FL 19 ml / (hu-Flt3L-Ig M. Manz, Üniversite tarafından sağlanan bir ekspresyon sistemi kullanılarak in-house olarak üretilmiştirty Hastanesi Zürich, İsviçre) ve 37 o C, mikroskop altında DC gelişme periyodik görsel izleme ile% 5 CO 2 inkübe. Not: Piyasada mevcut rekombinant insan FL veya fare FL DC gelişimini desteklemek için hu-Flt3L-Ig için bir yedek olarak kullanılabilir. </li><li> 12. günde süpernatan hücreleri toplamak ve elde edilen süpernatantlar birleştiren tam RPMI ortamına 0.5 ml ile bir kez kuyu yıkayın. Bir hücre kazıyıcı ile yapışık hücrelere 0.5 mL taze orta ekleyin ve hurda. </li><li> 500 x g&#39;de 5 dakika boyunca hücre içeren her iki parçadan orta ve santrifüj birleştirin. </li><li> 50 ul FACS tampon Durusu süpernatant ve tekrar süspansiyon hücreleri. 50 ul anti-CD16 / 32 hibridom süpernatant ekleyin ve Fc reseptörleri bloke etmek için 1-2 dakika inkübe edilir. </li><li> Numaralandırmak ve müteakip antikorlar (0,05 ug her biri), anti-CD11c-APC (N418), anti-CD11b-FITC, anti-B220 ve-PE tüm hücreleri leke. </li><li> Tarif olarak yıkayın ve santrifüj hücreleriadım 3.3, d. </li><li> CD11c + 100 ul FACS tamponu kapısı hücrelerinin yeniden süspanse ve CDCS (CD11c + CD11b + B220 -) için analiz ve pDC&#39;lerde (CD11c + CD11b - B220 +) 17. </li></ol>

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Authors have nothing to disclose.

Burada CLPs küçük bir sayı, özellikle bir in vitro kültür DClerin sağlam oluşturulması için bir sistem ve PDC tarif eder. Bu kültür sisteminin benzersiz besleyici olarak AC-6 hücreleri, stromal hücre çizgisi, kullanımına bağlıdır. Bu yaklaşım, IL-7, SCF, M-CSF ve FL 20 gibi yalnızca sitokinleri sağladığı gösterilmiştir, ancak, aynı zamanda, gerekli hücre-hücre teması 21 DC gelişimini desteklemek. AC-6 hücreler, birkaç progenitörlerin 8,16,22...

Dendritik hücreler (DC) bağışıklık tepkilerini 1 kontrolünde önemli bir rol oynamaktadır profesyonel antijen sunan hücrelerdir. DH&#39;ler, heterojen olsa da, genel olarak iki popülasyon olarak sınıflandırılabilir, konvansiyonel DCler (CDCS) ve plazmasitoid DCler (PDC) 2,3. Lenfoid organlara ek olarak, CDCS ve PDC aynı zamanda, akciğer, bağırsak, deri 2 dahil olmak üzere dokuların bulunurlar. CDCS ve pDC&#39;lerde morfolojisi dendrit gibi projeksiyonlar ve daha plazma hücre benzeri olma pDC&#39;lerde şeklini gösteren CDCS ile farklıdır. Buna ek olarak, ortak bir fare DC markör, CD11c, daha çok pDC&#39;lerde göre daha CDCS üzerinde ifade edilir. PDC 2 için, MHC sınıf II yüksek seviyeleri ve kostimülatör moleküller, her ikisi de CD24 + CDCS, - CDCS ve CD11b - Ayrıca, CDCS daha CD11b + CD24 ayrılabilir. Olgun PDC, diğer yandan, selektif Siglec-H ve BST2 4 ifade ettiği gösterilmiştir. Functionally, CDCS PDC olduğundan daha sunan hücrelerin iyi antijen; Ancak, PDC Ben virüs enfeksiyonu veya enflamatuar stimülasyon 5 üzerine interferon türü geniş bir miktarda üretebilir. Her iki CDCS ve PDC kısa ömürlü olduğu ve bu nedenle, sürekli kemik iliği (BM) 6,7 içindeki progenitörlerinden doldurulan olmalıdır. Öldürücü ışınlanmış farelere sık miyeloid progenitör (cmps) ve ortak lenfoid progenitörleri (CLPs) adoptif nakli CDCS ve PDC her iki soy 8-10 elde edilebileceğini göstermektedir. Ancak, RAG1 / 2 ifade ve IgH lokus 11,12 olarak yeniden düzenlenmiş DJ kesimleri sahip pDC&#39;lerde bir alt kümesi vardır. Bu hücreler, aynı zamanda B220 işaretleyicinin ifadesi, bir nükleik asit-algılama reseptörleri (TLR7 / TLR9) ve transkripsiyon faktörlerinin (SPIB ve Bcl11a) 13 de dahil olmak üzere B limfositleri üzerinde, moleküler benzerlikler, lenfoid imzaları olduğuna inanılmaktadır tüm sahiptir. Bu nedenle, CLPs nedeniyle soy benzerlik pDC&#39;lerde in vitro nesil için iyi seçimler olabilir. İnsanlarda ve farelerde hem CDCS ve pDC&#39;lerde sıklığı çok düşük olsa da 6 DH&#39;ler, özellikle de CDCS (örneğin, GM-CSF ya da 11,14 Flt3 ligandı gibi sitokinlerin mevcudiyetinde BM ya da progenitörlerinden in vitro üretilebilir FL ) besleyici içermeyen sistemler kullanılarak 11,15,16. Ancak ne yazık ki, bu FL 11,15,16 kullanılarak in vitro pDC&#39;lerde sayıda üretilmesi mümkün değildir. Daha önce PDC verimli AC-6 besleme sistemi 17 kullanarak CLPs in vitro olarak üretilebilir olduğunu gösterdi. Kültür sisteminde AC-6 stromal hücre çizgisi kullanılarak avantajı, hücre-hücre temas ve CLPs gelen pDC&#39;lerde çok sayıda üretilmesini destekleyen sitokinlerin salgılanmasını sağlamasıdır. Bu sistem ile, üretim oldukça sağlamdır olmasına rağmen, aşağıda anlatılan prosedürlerin dikkat çoğaltma i gereklidirn emri pDC&#39;lerde verimli nesil sağlamak.

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

Plazmasitoid dendritik hücreler (PDC) I enfeksiyona veya enflamatuar yanıtlar sırasmda yanıt olarak aktive olmuş (IFN-I) üreten hücreler interferon güçlü bir tipidir. Ne yazık ki, pDC fonksiyonunun çalışma lenfoid organlarda düşük frekansta tarafından engellenmektedir, in vitro DC nesil için mevcut yöntemler ağırlıklı pDC&#39;lerde üzerinde CDCS üretimini tercih. İşte biz verimli in vitro ortak lenfoid atalarıdır (CLPs) den PDC oluşturmak için benzersiz bir yaklaşım sunuyoruz. Spesifik olarak, iletişim kuralı, kemik iliğinden CLPs arındırmak ve Flt3 ligand mevcudiyetinde γ-ışınlanmış AC-6 besleyici hücreler ile coculturing ile PDC oluşturmak için nasıl ayrıntıları açıklanan. Bu kültür sisteminin benzersiz özelliği CLPs AC-6 hücreleri altında göç ve Arnavut kaldırımlı alan oluşturan hücreler, PDC genişletilmesi için kritik bir adım haline olmasıdır. Morfolojik olarak farklı DCler, yani PDC ve CDCS 7, bir bileşim ile yaklaşık 2 hafta sonra elde edildiOptimal koşullarda% 0-90 PDC. Tipik olarak, bu yöntemle üretilen pDC&#39;lerde sayısı kabaca tohumlu CLPs sayısı 100 mislidir. Bu nedenle, bu sağlam bu hücrelerin gelişimi ve fonksiyonu içine daha ileri çalışmalar kolaylaştırmak için gereklidir pDC&#39;lerde sayıda üretmek için hangi yeni bir sistemdir.

4-6x10 7 BM hücrelerinin toplam tipik haliyle femur ve bir 6-8 hafta yaşında, vahşi tipli C57BL / 6 fare tibya izole edilmiştir. CLPs sıralamak için, toplam BM hücrelerinin soy belirteçleri (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119 ve MHC-II), anti karşı PE-konjuge antikor ile boyanır c-Kit PerCP / Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC, anti-IL-7Rα-PE / Cy7, analiz edilir ve bir hücre sınıflandıncısı ile kriteri. CLP için sıralama strateji Şekil <strong...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6 Besleyici Sistemi kullanılarak Ortak Lenfoid progenitörlerin dan Fare plazmositoid dendritik hücreler İn Vitro Nesil

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.3K Görüntüleme.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

Video: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

İzleme Araştırmaları Qdot nanokristaller murin Kemik İliği-türevli dendritik hücrelerin üretilmesi ve Etiketleme

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

İmmünoloji ve Enfeksiyon

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.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

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 ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Melanom ve Skuamöz Kanser Kök Hücreleri Kullanılarak Yeni Bir Dendritik Hücre aşısının Üretilmesi

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Fare Kemik İliği itibaren Üretimi ve GM-CSF tanımlanması Türetilmiş Alveoler gibi makrofajlar ve dendritik hücrelerin

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fare kemik iliğinden plazmasitoid dendritik hücreleri Saflaştırılması floresans ile aktive edilen hücre sınıflandırma

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Tüm kan İnsan monosit türevli dendritik hücrelerin oluşumu

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Çok sayıda miyeloid ataları ve dendritik hücre öncüleri fare kemik iliği strateji sıralama bir roman hücre kullanarak gelen nesil

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...