PMID- 20875421 OWN - NLM STAT- MEDLINE DCOM- 20101230 LR - 20231213 IS - 1872-7905 (Electronic) IS - 0022-1759 (Print) IS - 0022-1759 (Linking) VI - 363 IP - 1 DP - 2010 Dec 15 TI - Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation. PG - 21-8 LID - 10.1016/j.jim.2010.09.028 [doi] AB - 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 naive 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 IFNbeta mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs. CI - Copyright (c) 2010 Elsevier B.V. All rights reserved. FAU - Bowles, Robert AU - Bowles R AD - 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 FAU - Patil, Sonali AU - Patil S FAU - Pincas, Hanna AU - Pincas H FAU - Sealfon, Stuart C AU - Sealfon SC LA - eng GR - N01 AI050021/AI/NIAID NIH HHS/United States GR - HHSN2662000500021C/PHS HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20100924 PL - Netherlands TA - J Immunol Methods JT - Journal of immunological methods JID - 1305440 RN - 0 (B7-2 Antigen) RN - 0 (CD86 protein, human) RN - 0 (Histocompatibility Antigens Class II) RN - 0 (RNA, Small Interfering) RN - 0 (Receptors, Immunologic) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 77238-31-4 (Interferon-beta) RN - EC 3.6.1.- (RIGI protein, human) RN - EC 3.6.4.13 (DEAD Box Protein 58) RN - EC 3.6.4.13 (DEAD-box RNA Helicases) SB - IM MH - B7-2 Antigen/biosynthesis/genetics MH - DEAD Box Protein 58 MH - DEAD-box RNA Helicases/biosynthesis/genetics MH - *Dendritic Cells/cytology/metabolism MH - Flow Cytometry/methods MH - Gene Expression MH - Gene Knockdown Techniques/*methods MH - Green Fluorescent Proteins/biosynthesis/genetics MH - Histocompatibility Antigens Class II/biosynthesis/genetics MH - Humans MH - Interferon-beta/biosynthesis/genetics MH - *Monocytes/cytology/metabolism MH - Newcastle disease virus/genetics/metabolism MH - *Plasmids MH - RNA Interference MH - *RNA, Small Interfering MH - Receptors, Immunologic MH - Transfection/*methods PMC - PMC3964480 MID - NIHMS245874 EDAT- 2010/09/30 06:00 MHDA- 2010/12/31 06:00 PMCR- 2014/03/25 CRDT- 2010/09/30 06:00 PHST- 2010/09/14 00:00 [received] PHST- 2010/09/17 00:00 [accepted] PHST- 2010/09/30 06:00 [entrez] PHST- 2010/09/30 06:00 [pubmed] PHST- 2010/12/31 06:00 [medline] PHST- 2014/03/25 00:00 [pmc-release] AID - S0022-1759(10)00282-6 [pii] AID - 10.1016/j.jim.2010.09.028 [doi] PST - ppublish SO - J Immunol Methods. 2010 Dec 15;363(1):21-8. doi: 10.1016/j.jim.2010.09.028. Epub 2010 Sep 24.