Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Summary

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.

Abstract

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 naïve 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^2,3. 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 DCs^9,10,11,12. Electroporation has been used with mixed results^13,14,15, 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 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^16,17, at both the mRNA and protein levels.

Protocol

1. Program the Amaxa 96 well shuttle Nucleofector

1. Open a new parameter file.
2. 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.
3. Input the program code: in part1 select 'FF' and in part2 select '168' from the pull down menus
4. From Solution box select 'Monocyte, human'
5. Under Control Option select 'standard'.
6. Click on Apply.
7. To include a no-transfection control, select further wells from the diagram as required and then choose 'No Program Control' from Control Opt....

Discussion

Efficient transfection of naïve 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amaxa Nucleofector 96-well Shuttle	Lonza Inc.	108S0109	Serial number
Amaxa Human Monocyte 96-well Nucleofector Kit	Lonza Inc.	VHPA-2007	Contains the Human Monocyte 96-well Nucleofector Solution, the 96-well Supplement and the Nucleocuvettes and plates
RIG-I siRNA	Dharmacon	L-012511-00
GLO siRNA	Dharmacon	D-001600-01-20
RPMI 1640	Invitrogen	11875	Supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin to make DC growth medium
DMEM	Invitrogen	11965
L-glutamine	Invitrogen	25030081
Penicillin/streptomycin	Invitrogen	15070063
Fetal Calf Serum	Hyclone	3070.03
Dendritic Cells	New York Blood center		DCs are purified from buffy coats using a standard procedure