Yazarlar bu yazının verileri katkıda Michael Chou, Bingbing Dai ve Liang Xiao kabul etmek istiyorum. Biz de bu çalışmada kullanılan reaktiflerin cömert hediyeler için Dr John Gray kabul. PB Ulusal Kanser Merkezi&#39;nden bir doktora sonrası burs tarafından desteklenmektedir. Bu araştırma Ulusal Sağlık Enstitüsü (R01AI68978, P01CA132681 ve RCA170820A), Bill ve Melinda Gates Vakfı, Translational Medicine için Ortak Merkezi&#39;nden bir öteleme ivme hibe ve Kaliforniya HIV / AIDS bir hibe bir hibe hibe tarafından desteklenmiştir Araştırma Programı.

DC-LV istikrarlı üretici hücreleri gerekli lentiviral bileşenleri gagpol, devir ve tet-off sistemi içeren GPR ambalaj hücre hattı 11 temel inşa edilir. İlk olarak, bir retroviral transdüksiyon tet-bağımlı SVGmu glikoprotein kodlayan bir GPRS paketleme hücre hattı üretmek için kullanılır. Daha sonra, bir dizi concatemer transfeksiyon gibi GFP gibi bir lentiviral vektör transgen ile GPRS hücre hattı transfekte etmek için kullanılır. AG-MGFP olarak tanımlanan bu stabil üretici hücre hattı, GFP karşı, bir DC-AG aşı üretme kabiliyetini için in vitro ve in vivo olarak test edilebilir. 1. Bir Tet bağımlı SVGmu Hücre Hattı oluşturma Plazmid PrX-SVGmu DC-özgü glikoproteini SVGmu alt retroviral plazmid pRetroX-Tet fiyatına 1 (Şekil 1C) tTA-gelişmiş promotör klonlanmış olduğu bir yapıdır. D10 Kültür 293T hücreleri (Dulbecco değiştirilmiş Eagle ortamı ile% 10 fetal bovin serumu, 2 mM L-glutamin). Doksisiklin (1 ng / ml) ve puromisin (2 ug / ml) ile D10 Kültür GPR paketleme hücre hattı. Tüm hücreler% 5 CO2 ile nemlendirilmiş 37 ° C inkübatör içinde kültürlendi. <ol><li> 16-18 saat önce, geçici transfeksiyon, levha 2 x 10 hücre transfeksiyon zaman az% 90 konfluent yaklaşım, böyle bir 6-cm doku kültürü çanağı içinde D10 4 ml 6 293T hücreleri. </li><li> Retroviral plasmidlerin transfeksiyonu: karışım 100 ul 1.25 M CaCI2 çözeltisi steril Milli-Q su ve aşağıdaki plazmid: 5 ug PrX-SVGmu, 2.5 ug pGag-Pol, 2.5 ug pVSV-G. Son hacim 500 ul. 5 ml polistiren yuvarlak tabanlı tüp, 500 ul 2X HBS (50 mM HEPES, 10 mM KCI, 12 mM Dekstroz, 280 mM NaCI, 2 HPO 4 * 7H 2, O, pH 7.05, 1.5 mM Na) ekleyin. Bir cam Pasteur pipeti ile şiddetli bir şekilde tampon köpüren ise 2X HBS tampon plazmid karışımı damla damla ilave edin. Mi ekledikten sonraxture, bir 30 saniye boyunca köpüren devam. Daha sonra, 6 cm kültür tabağına 293T hücreleri üzerine bütün karışımı eklenir, ve 37 ° C de inkübe </li><li> 4 saat transfeksiyon sonra, dikkatli bir şekilde 4 ml önceden ısıtılmış D10 ile kültür çanak orta yerine. </li><li> Enfeksiyon Spin: Transfeksiyondan 48 saat sonra hasat SVGmu-kodlayan bir 0.45 mikron filtre içinden geçen süpernatant ile süpernatantı retroviral parçacıklar. / Iyi 2 x 10 4 hücre bir 24-iyi çanak plaka GPR ambalaj hücreleri. 1,050 x g&#39;de 90 dakika ve 25 ° C için çanak GPR paket hücreleri (2 ml / oyuk) ve santrifüj hücrelere Süpernatan süzülmüş ekleme (2 ml / oyuk) Spin-enfeksiyon sonrası doksisiklin (1 ng / ml) ile taze D10 içine orta değiştirin. </li><li> Paket hücreleri doksisiklin (1 ng / ml) ve puromisin (2 ng / ml) ile D10 transduse 72 saat post-transfeksiyon, kültür genişletmek. </li><li> 48 saat doksisiklin olmadan SVGmu, kültür hücrelerin ifade onaylamak için. Yüzey eski ölçünAnti-Sindbis serum kullanılarak akım sitometri ile SVGmu bir depresyon. Bu hücreler, GPRS, paketleme hücre olarak adlandırılır. </li></ol> 2. Concatemer Dizi Transfeksiyon tarafından DC-LV Üretici Hücreler Construct Lentiviral transferi TL20 plazmid GFP bir kendi kendine inaktif lentiviral transfer vektörü bir plazmiddir Dox-regüle viral bir RNA genomu ekspresyon sistemi ve 7 tet operatör (Şekil 1C) 11, sitomegalovirüs (CMV) güçlendirici yer değiştirmesi ile pCL20c-MSCV-GFP dayalı , 12,15. Plazmid PGK-ble zayıf bir PGK organizatörü 2 tarafından yönlendirilen bir bleomisin dayanıklı (dür) kaset olduğunu. <ol><li> 50 kısıtlama enzimi sindirimi Sfil ile 20 ug plazmid TL20-GFP ° C 37 PflMI ile Digest 20 mg plazmid PGK-ble ° C </li><li> Agaroz jel elektroforezi ile DNA fragmanları (PGK-labilir kaseti 1011 bp ve vektör TL20-GFP 6861 bp) arıtınız. </li><li> TL20-GFP vektör ve PGK-Arter25:1 &#39;lik bir molar oranda (NEB, T4 DNA ligaz) de ble kaseti. Gece boyunca oda sıcaklığında inkübe edin. </li><li> Gece boyunca ligasyon sonra ligasyon karışımı (Qiagen DNeasy Kan ve doku Kiti) DNA arındırmak. Arıtılmış DNA miktarını sağlamak 5 mg civarındadır. </li><li> Transfeksiyondan 16-18 saat önce, konfluent transfeksiyon zaman yaklaşık olarak% 90 olacak şekilde, 6 cm doku kültürü çanağı içinde plaka GPRS paket hücreleri. </li><li> 6 cm doku kültürü çanağı içinde GPRS paket hücreleri içine concatemeric saflaştırılmış DNA 5 ug transfekte etmek için aşama 1.2 &#39;de tarif edilen kalsiyum fosfat transfeksiyon yöntemi. Transfeksiyon, 4 saat sonra, dikkatli bir şekilde orta kaldırmak ve doksisiklin (1 ng / ml) ile 4 ml önceden ısıtılmış D10 ile değiştirin. </li><li> Transfeksiyondan 48 saat sonra, doksisiklin (1 ng / ml) ve puromisin (2 ug / ml) ile D10 içine orta değiştirmek. Kültür ortamı içinde zeosin (50 mg / ml) eklenerek transfekte klonlar için seçin. Hücre koloni kadar yaklaşık 2 hafta boyunca kültür hücreleries yemekleri altındaki görülebilir. </li><li> Yemeklerin altındaki hücre kolonileri etiketleyin. , 24 oyuklu doku kültürü tabaklarında al sayıda kuyu ve her biri de 2 ml zeosin D10 içeren (50 ug / ml), doksisiklin (1 ng / ml) ve puromisin (2 ug / ml) ekleyin. Transduse edilmiş hücrelerin medyumu aspire, o zaman en az bir dakika süre ile kolonilerin her birisi üzerine tripsin bir damla ilave edin. Daha sonra, aynı koloniler D10, bir ya da daha fazla damla ilave edin. Bir pipet yardımıyla tek koloniler bir pick up ve 24-iyi doku kültürü plakası ayrı kuyu içine aktarın. </li><li> Kültür ve viral üreten yeteneğinin değerlendirilmesi için zeosin D10 içeren (50 ug / ml), doksisiklin (1 ng / ml) ve puromisin (2 ug / ml) içindeki tüm hücre klonları genişletebilir. </li></ol> 3. Her Hücre Clone Viral Üretim değerlendirin <ol><li> Doksisiklin olmadan birbirine karışmış gibi D10, 6 cm doku kültürü çanağı içinde 4x10 6 hücre etrafındaki üretici hücreler ve levha Trypsinize% 90 aşıyor. Günlük taze önceden ısıtılmış D10 ile orta değiştirin ve dox çıkardıktan sonra titre testi 72 saat için orta toplamak. </li><li> Bir 0.45 mikron filtre ile filtreleme ortamı ile viral süpernatan hasat. </li><li> Plaka hedef hücreler 4 x 10 hücre / 1, 96-çukurlu kültür tabaklarında bir negatif kontrol olarak, DC-SIGN reseptörü (örneğin, elde edilen 293T.hDC-SIGN veya fare kemik iliği dendritik hücreler 16) ve 293T ifade. Kuyu (göz başına 100 ul /) için viral üstte yüzen 2-kat seri dilüsyonları ekleyin. Genellikle, 6-8 seyreltileri GFP pozitif hücreleri ilave virüs miktarı için doğrusal bir ilişki içinde olan transduse hangi aralığı ulaşmak için yeterlidir. </li><li> Hücreler santrifüj ve benzeri gibi Adım 1.4 &#39;de tarif edilen orta yerine. BMDCs% 10 FBS ve GM-CSF (1:20 J558L koşullandırılmış ortam) ile RPMI ortamında edilmelidir. </li><li> Akım sitometri 4-6 gün sonrası transdüksiyon hücreleri transduced en GFP ifade ölçün. Lentiviral vectoGFP-pozitif hücreler ve vektör miktarının yüzde doğrusal bir ilişki içinde olduğunda R titresi vektör seyreltme aralığında hesaplanır. Sonraki uygulamalar için en yüksek virüs üretimi ile hücre klonu seç. </li></ol> 4. Lentiviral Vektörler üretmek ve Konsantre <ol><li> Kültür 15 cm doku kültür tabaklarında üretici hücre hattı (AG-MGFP). </li><li> Doksisiklin olmadan taze D10 konfluense% 90 daha az 15 cm doku kültürü yemekleri üretici hücreleri ve plaka hücreleri Trypsinize. Günlük taze, önceden ısıtılmış D10 orta değiştirin. </li><li> Pik viral üretimi (kullanıcı tarafından tanımlanır) bir zamanda, viral süpernatan hasat ve bir 0.45 mikron filtre kullanılarak filtre. 90 dakika boyunca 50,000 x g&#39;de santrifüj parafin ve, 4 ° C mühür, kalın duvar 32.5 ml&#39;lik bir ultra santrifüj tüpleri içine süzüldü süpernatant yerleştirin. İyice 50 ul veya uygulamaya bağlı olarak, PBS veya HBSS uygun bir hacmi pelletini. &lt;/ Li&gt; </ol> 5. In vivo farelerine bağışıklık ve antijen-spesifik bir bağışıklık tepkisi Analiz <ol><li> Bölüm 4&#39;te açıklandığı gibi yüksek titre virüs üretin. </li><li> Vektör süspansiyon (ayak yastığı başına 25 ul) ile bir haydut yolla BALB / c farelerde (eski, kadın 6-8 hafta) enjekte edilir. </li><li> 2 hafta aşılama sonra, dalak ve hasat dalak izole. GFP epitop peptitleri ile ve GFP-özgül CD8 + varlığında, hücre içi sitokin boyaması kullanılarak bir T-hücrelerinde analiz Kültür splenositler, daha önce 17 (Şekil 3B) tanımlamışlardır. </li></ol>

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<p class="jove_content"&gt; Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.</p

Burada, stabil olarak düzenleyici sistem kapalı Tet altında lentiviral bileşenleri ile transduse 293T hücreleri kullanılarak lentiviral vektörleri büyük miktarlarda üretilmesi için bir yöntem, ana hatlarıyla. Bugüne kadar, lentiviral vektörleri üretmek için en çok kullanılan protokol (örneğin, 18), standart kalsiyum fosfat geçici transfeksiyon dayanmaktadır. Bu yaklaşım bir klinik ölçekte başarılı olmuştur, ama istikrarlı hücre hatları üretimi ölçeklendirme mevcut olması ...

Birçok vektör sistemleri gen aktarımı için geliştirilmiştir. Lentivirüsler dayalı vektörler en sık çalışılan viral sistemi arasında yer almıştır. Verimli bir şekilde, hücreleri 1 bölme ve olmayan bölünmesi hem transduce nedeniyle ev sahibi genomuna entegrasyonu için uzun vadeli ifade elde, en insan nüfusu 2 düşük doğal anti-vektör bağışıklık sergi ve için düşük potansiyeline sahip, çünkü bu vektörler avantajlıdır girmeyle mutagenez 3,4 ikinci genotoksisite. Lentivirüsler üretimi her zaman güvenlik kaygıları ile renklendi. Lentiviral vektörleri, genellikle, HİV-1, AIDS etken ajan elde edilmektedir. Lentivector tek tek bileşenleri (transfer, zarf ve ambalaj plazmid) geçici transfeksiyon laboratuvar ortamlarında genetik materyal sunma ortak ve esnek bir araçtır. Ancak, klinik uygulamalar için ölçekleme-up geçici transfections hantal ve may çoğaltma yetkili lentivirüs 5,6 gelişmesine yol açar. Bu engellerin üstesinden gelmek için, çeşitli kararlı ambalaj ve üretici hücre çizgileri 6-11 geliştirilmiştir. Bu hatlarından biri, GPR paketleme hattı 11, tetrasiklin tarafından düzenlenen olma cazip bir avantaja sahiptir. Bu yazıda, özellikle dendritik hücreler (DC-LVs) 12 doğru hedeflenmektedir kendini inaktive lentiviral vektörler üretmek için bu sistem uyum nasıl gösterilmektedir. Dendritik hücreler (DC) bağışıklık sisteminin en güçlü antijen sunan hücrelerdir. Doğrudan, programı başlatmak ve tümör spesifik immün yanıtları 13 düzenleyen çünkü kanser aşısı geliştirilmesi büyük ilgi konusu olmuştur. DC peptid veya DNA aşıları daha güçlü bir anti-tümör immün yanıt ortaya çıkarmak potansiyeline sahip dahil etmek için bir aşı protokolü içeren. Son zamanlarda, bir lentiviral vektör geliştirdiğimiz specifically, bir modifiye sindbis virüsü glikoprotein, SVGmu 14 boyunca dendritik hücreler hedeflemektedir. Onlar dendritik hücreler için yüksek özgüllük göstermek ve spesifik olmayan, VSVg-pseudotyped vektörler daha güçlü bağışıklık yanıtları üreten bu vektörler benzersizdir. Burada, bu DC-hedefli lentiviral vektörleri büyük miktarlarda üretilmesi için bir yöntem sunulmuştur. Biz bu DC-DC LVs bulaştırmak ve güçlü CD8 + T hücre bağışıklık yanıtı oluşturabilir göstermektedir. Hayvanlar ile ilgili tüm işlemler USC Intitutional Hayvan Bakım ve Kullanım Kurulu onayı altında, insanca yapıldı. In vivo ve klinik deneylerde gerçekleştirmek için, bu yüksek bir titresi virüs üretebilen bir hücre çizgisi oluşturmak için çok önemlidir. Aynen anlatıldığı gibi iletimi ve transfeksiyon adımları uygulayarak böyle bir klon üretme şansını maksimize edecek.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Lentiviral vektörleri (karaciğer hacimleri) hücrelerin birçok çeşit genetik materyal teslim güçlü araçlardır. LVS büyük miktarlarda üretim, bu, HIV-1 ile ilişkili elde edilen vektörler, güvenlik kaygıları nedeniyle zordur. Bu yazıda, kendi kendini inaktive karaciğer hacimleri yüksek titreleri üretmek için bir yöntem rapor. Bu retroviral olarak bir dendritik hücre-özgü glikoproteini, SVGmu de dahil olmak üzere, tüm viral bileşenler ifade edebilen bir hücre hattı, GPRS oluşturmak için tet fiyatına stabil üretici hücre hattı GPR iletmektedir. Daha sonra, sol ventrikül aktarım plazmid kodlayan bir seçilebilir marker ile bir arada bir GFP raportör gen transfekte etmek için concatemeric DNA transfeksiyon kullanın. Sonuçta elde edilen klonlar çeşitli biz geçici transfeksiyon ile elde olandan 10 kat daha fazla bir titre de LV üretebilir. Ayrıca, bu virüsler, in vitro olarak verimli bir şekilde dendritik hücrelerin transdüksiyonu ve muhabir antijene karşı güçlü bir T hücresi bağışıklık tepkisi oluşturur. Bu yöntem iyi bir seçenek olabilir for kanser veya bulaşıcı hastalıkların klinik çalışmalar için güçlü LV-tabanlı aşısı üreten.

Bu yöntem, anlatılan stabil hücre hattı, özellikle dendritik hücreler için hedeflenen lentiviral vektörleri büyük miktarda üretebilir. Şekil 1B &#39;de gösterildiği üzere, bireysel klonların izolasyonu kaliteli 12 arasında değişen stabil hücre hatları elde edilmiştir. Test edilmiş 26 klon içinde, 8 geçici transfeksiyon tarafından üretilen SVG pseudotyped lentiviral vektörleri için tipik bir ölçüttür ml (TU / ml) başına 10&#39;dan daha fazla 6 biri...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Burada, retroviral transdüksiyon ve tetrasiklin yokluğunda bir lentiviral vektör (IV) bileşenleri ifade edebilen bir hücre çizgisi oluşturmak için concatemeric transfeksiyon kullanın. Bu AG GFP kodlar ve dendritik hücreler üzerinde reseptörü için spesifik olan bir glikoprotein, SVGmu ile pseudotyped edilir.

Bir Tetrasiklin düzenlenmiş Hücre Hattı Özellikle dendritik hücreler Hedef Yüksek titre Lentiviral vektörler üretir

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K Görüntüleme.  University of Southern California, Los Angeles. Burada, retroviral transdüksiyon ve tetrasiklin yokluğunda bir lentiviral vektör (IV) bileşenleri ifade edebilen bir hücre çizgisi oluşturmak için concatemeric transfeksiyon kullanın. Bu AG GFP kodlar ve dendritik hücreler üzerinde reseptörü için spesifik olan bir glikoprotein, SVGmu ile pseudotyped edilir.

Video: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Hücre olgunlaşması olmadan Dendritik Hücreler Verimli Transfeksiyon için optimize edilmiş Protokolü

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

İmmünoloji ve Enfeksiyon

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Fare Akciğer Dendritik Hücrelerinin İzolasyonu

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

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

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

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

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

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Kullanarak İzleme Dendritik Hücre Göç<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Manyetik Rezonans Görüntüleme

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 ...

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 ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Metabolik fenotipleri farklı olarak sahip Olgunlaşmamış, Olgun ve tolerojenik Dendritik Hücrelerinin Üretimi

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Escherichia Coli proteinler Site-Specifically oluşturmak için Facile iletişim kuralı Acetylated

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Geliştirme ve doğrulama vericiyi bir tek molekül dijital enzim bağlı Immunosorbent Assay insan Interferon-α için dizi

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...