The project was supported by NIH NIAID Contract No. HHSN2662000500021C. We thank Ming Chen for his technical assistance.

1. Program the Amaxa 96 well shuttle Nucleofector 
<ol>
 <li>Open a new parameter file.</li>
 <li>Select the number of wells you will be using for standard transfection by dragging the cursor over the 96 well plate diagram. Use a minimum of 3 wells to pool for each experimental sample.</li>
 <li>Input the program code: in part1 select 'FF' and in part2 select '168' from the pull down menus</li>
 <li>From Solution box select 'Monocyte, human'</li>
 <li>Under Control Option select 'standard'. </li>
 <li>Click on Apply.</...

<ol>
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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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No conflicts of interest declared.

Efficient transfection of na&iuml;ve primary dendritic cells is important for high throughput analysis and reverse engineering of cellular inflammatory pathways in this key cell mediating the innate-adaptive immune transition. However, most investigators find that these cells are difficult to transfect both efficiently and without the transfection procedure inducing cell maturation when using standard transfection techniques. We investigated whether these limitations could be overcome by high throughput protocol optimiza...

<table border="1">
	<tbody>
		<tr>
			<th>Equipment/Reagent</th>
			<th>Company</th>
			<th>Catalogue #</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Amaxa Nucleofector 96-well Shuttle</td>
			<td>Lonza</td>
			<td>108S0109</td>
			<td>Serial number</td>
		</tr>
		<tr>
			<td>Amaxa Human Monocyte 96-well Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VHPA-2007</td>
			<td>Contains the Human Monocyte 96-well Nucleofector Solution, the 96-well Supplement and the Nucleocuvettes and plates</td>
		</tr>
		<tr>
			<td>RIG-I siRNA</td>
			<td>Dharmacon</td>
			<td>L-012511-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GLO siRNA</td>
			<td>Dharmacon</td>
			<td>D-001600-01-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td>Supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 &mu;g/ml streptomycin to make DC growth medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>HyClone</td>
			<td>3070.03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dendritic Cells</td>
			<td>New York Blood center</td>
			<td>&nbsp;</td>
			<td>DCs are purified from buffy coats using a standard procedure</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

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.

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

optimized-protocol-for-efficient-transfection-dendritic-cells-without

Research

JoVE Journal

Immunology and Infection

20.2K visualizações.  Mount Sinai School of Medicine. Apresentamos nosso protocolo nucleofection high-throughput otimizado como uma forma eficiente de transfecting humanos primários derivados de monócitos com células dendríticas ou DNA plasmídeo ou siRNA sem causar maturação das células. Nós ainda fornecer provas para silenciar siRNA sucesso do gene alvo RIG-I em ambos os mRNA e os níveis de proteína.