Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
| Autor: | Stuart C. Sealfon, Robert N. Bowles, Hanna Pincas, Sonali Patil |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
General Chemical Engineering
Green Fluorescent Proteins Immunology Nucleofection chemical and pharmacologic phenomena Cell Separation Biology Transfection General Biochemistry Genetics and Molecular Biology DEAD-box RNA Helicases Transduction (genetics) Immune system Humans RNA Small Interfering Receptors Immunologic CD86 General Immunology and Microbiology Electroporation General Neuroscience Pattern recognition receptor hemic and immune systems Dendritic Cells Interferon-beta Acquired immune system Flow Cytometry Cell biology Gene Knockdown Techniques DEAD Box Protein 58 Plasmids |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/2766-v |
| Popis: | Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection. Detection of pathogenic antigen by naive 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 response. 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 DCs. 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). Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs. Electroporation has been used with mixed results, 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 maturation. 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 IFNβ. 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 receptor, at both the mRNA and protein levels. |
| Databáze: | OpenAIRE |
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