title: Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation.
abstract: Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNβ mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.
authors.affiliation: Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. robert.bowles@mssm.edu
authors.forename: Robert
authors.initials: R
authors.lastname: Bowles
authors.forename: Sonali
authors.initials: S
authors.lastname: Patil
authors.forename: Hanna
authors.initials: H
authors.lastname: Pincas
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Sealfon
mesh_terms: D051940:B7-2 Antigen / Q000096:biosynthesis / Q000235:genetics; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000096:biosynthesis / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D009522:Newcastle disease virus / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D014162:Transfection / Q000379:methods*
chemical_list: D051940:B7-2 Antigen; C497964:CD86 protein, human; D000949:Histocompatibility Antigens Class II; D034741:RNA, Small Interfering; D011971:Receptors, Immunologic; D049452:Green Fluorescent Proteins; D016899:Interferon-beta; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases