title: Using RNA-interference to investigate the innate immune response in mouse macrophages.
abstract: Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.
authors.affiliation: Integrated Department of Immunology and Integrated Center for Genes, Environment, and Health, National Jewish Health and University of Colorado School of Medicine.
authors.forename: Lesly
authors.initials: L
authors.lastname: De Arras
authors.affiliation: Integrated Department of Immunology and Integrated Center for Genes, Environment, and Health, National Jewish Health and University of Colorado School of Medicine.
authors.forename: Brandon S
authors.initials: BS
authors.lastname: Guthrie
authors.affiliation: Integrated Department of Immunology and Integrated Center for Genes, Environment, and Health, National Jewish Health and University of Colorado School of Medicine; alpers@njhealth.org.
authors.forename: Scott
authors.initials: S
authors.lastname: Alper
mesh_terms: D000818:Animals; D002460:Cell Line; D018925:Chemokines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016207:Cytokines / Q000276:immunology; D055785:Gene Knockdown Techniques; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D000067996:RAW 264.7 Cells; D034622:RNA Interference / Q000276:immunology*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology; D014162:Transfection / Q000379:methods
chemical_list: D018925:Chemokines; D016207:Cytokines; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering