Name: optimized protocol for efficient transfection of dendritic cells without cell maturation
Uploaded: 2011-07-08T13:00:00
Description: Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

परियोजना NIH NIAID अनुबंध सं. HHSN2662000500021C द्वारा समर्थित किया गया. हम अपने तकनीकी सहायता के लिए मिंग चेन धन्यवाद.

1. कार्यक्रम Amaxa 96 अच्छी तरह शटल Nucleofector <ol><li> एक नए पैरामीटर फ़ाइल खोलें. </li><li> आप 96 अच्छी तरह से थाली आरेख पर कर्सर खींचकर मानक अभिकर्मक का उपयोग करने के लिए किया जाएगा कुओं की संख्या का चयन करें. पूल करने के लिए ?...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat Rev Genet. 4, 346-358 (2003).</li><li>Jooss, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+dendritic+cells+by+DNA+viral+vectors+directs+the+immune+response+to+transgene+products+in+muscle+fibers.">Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.</a> J. Virol. 72, 4212-4223 (1998).</li><li>Mitchell, D. 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Methods. , .</li><li>Kato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+involvement+of+RIG-I+in+antiviral+response.">Cell type-specific involvement of RIG-I in antiviral response.</a> Immunity. 23, 19-28 (2005).</li><li>Kato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+MDA5+and+RIG-I+helicases+in+the+recognition+of+RNA+viruses.">Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.</a> Nature. 441, 101-105 (2006).</li><li>Haller, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mx+GTPase+family+of+interferon-induced+antiviral+proteins.">The Mx GTPase family of interferon-induced antiviral proteins.</a> Microbes Infect. 9, 1636-1643 (2007).</li></ol>

भोले प्राथमिक वृक्ष के समान कोशिकाओं के कुशल अभिकर्मक उच्च throughput विश्लेषण और इस कुंजी सहज अनुकूली प्रतिरक्षा संक्रमण mediating कक्ष में सेलुलर भड़काऊ रास्ते के रिवर्स इंजीनियरिंग के लिए महत्वपूर्ण है. हाल?...

<table border="1"><tbody><tr><th> / उपकरण अभिकर्मक </th><th> कंपनी </th><th> कैटेलॉग # </th><th> टिप्पणियाँ </th></tr><tr><td> Amaxa Nucleofector 96 अच्छी तरह शटल </td><td> Lonza </td><td> 108S0109 </td><td> सीरियल नंबर </td></tr><tr><td> Amaxa मानव 96 ही Nucleofector किट monocyte </td><td> Lonza </td><td> VHPA-2007 </td><td> मानव monocyte 96 अच्छी तरह से Nucleofector समाधान, 96 अच्छी तरह अनुपूरक और Nucleocuvettes और प्लेट शामिल </td></tr><tr><td> RIG - मैं siRNA </td><td> Dharmacon </td><td> एल - ०,१२,५११ 00 </td><td></td></tr><tr><td> GLO siRNA </td><td> Dharmacon </td><td> डी--001600 01 20- </td><td></td></tr><tr><td> 1640 RPMI </td><td> Invitrogen </td><td> 11875 </td><td> FCS 10%, के साथ पूरक 2 एल glutamine मिमी, 100 / यू मिलीलीटर पेनिसिलिन और 100 / μg मिलीलीटर के लिए डीसी मध्यम विकास स्ट्रेप्टोमाइसिन </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965 </td><td></td></tr><tr><td> एल-glutamine </td><td> Invitrogen </td><td> 25030081 </td><td></td></tr><tr><td> पेनिसिलीन / स्ट्रेप्टोमाइसिन </td><td> Invitrogen </td><td> 15070063 </td><td></td></tr><tr><td> भ्रूण बछड़ा सीरम </td><td> HyClone </td><td> 3070.03 </td><td></td></tr><tr><td> वृक्ष के समान कोशिकाओं </td><td> न्यूयॉर्क रक्त केंद्र </td><td></td><td> DCs buffy कोट का उपयोग एक मानक प्रक्रिया से शुद्ध कर रहे हैं </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation at JoVE.com

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

हम या तो प्लाज्मिड या सेल परिपक्वता के कारण के बिना डीएनए siRNA साथ प्राथमिक मानव monocyte व्युत्पन्न वृक्ष के समान कोशिकाओं transfecting की एक कारगर तरीका के रूप में हमारे अनुकूलित nucleofection उच्च throughput प्रोटोकॉल उपस्थित थे. हम आगे लक्षित जीन RIG मैं दोनों mRNA और प्रोटीन के स्तर पर सफल siRNA मुंह बंद करने के लिए सबूत प्रदान करते हैं.

सेल परिपक्वता के बिना वृक्ष के समान कोशिकाओं के कुशल अभिकर्मक के लिए अनुकूलित प्रोटोकॉल

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

Research

JoVE Journal

Immunology and Infection

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

जनरेशन और ट्रैकिंग अध्ययन के लिए Qdot Nanocrystals साथ murine अस्थि मज्जा व्युत्पन्न वृक्ष के समान कोशिकाओं के लेबल

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Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Immunotherapy के लिए ट्यूमर एंटीजन से भरी हुई परिपक्व वृक्ष के समान कोशिकाओं की तैयारी

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

कुशल के लिए खूनी कृत्रिम एंटीजन पेश कोशिकाओं (KaAPC)<em&gt; इन विट्रो</em&gt; मानव प्रतिजन विशेष टी कोशिकाओं की कमी

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Nucleofection द्वारा मानव THP-1 मैक्रोफेज की अत्यधिक कुशल अभिकर्मक

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

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माउस वृक्ष के समान सेल सबसेट के अलगाव के लिए एक कुशल और उच्च उपज विधि

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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

मेटाबोलिक phenotypes भिन्न के साथ अपरिपक्व, परिपक्व और Tolerogenic वृक्ष के समान कोशिकाओं की पीढ़ी

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

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फिजियोलॉजिकल इम्यूनोजेनिक डेन्ड्रिटिक कोशिकाओं के सृजन के लिए उपन्यास प्रोटोकॉल

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

समृद्ध मानव स्मृति बी सेल आबादी से वांछित विशिष्टताओं के साथ एंटीबॉडी के चयन और वसूली के लिए एकल सेल स्क्रीनिंग विधि

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...