title: Using Image-Based Flow Cytometry with a FISH-Based FlowRNA Assay to Simultaneously Detect Intracellular TNF-α Protein and mRNA in Monocytes Following LPS Stimulation.
abstract: Existing methods of assessing monocyte inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) response to in vitro lipopolysaccharide (LPS) stimulation lack the ability to simultaneously detect intracellular mRNA and protein. This procedure takes advantage of new methodologies and instrumentation to simultaneously measure intracellular TNF-α mRNA and protein in CD14(+) monocytes after 1, 3, and 6 h of LPS stimulation. By assessing multiple timepoints, we are able to discern how LPS stimulation affects the temporal relationship between TNF-α mRNA and protein. By using image-based flow cytometry it is possible to co-localize mRNA and protein signals to identify the length of incubation that is needed to initiate protein translation.
authors.affiliation: Applied Physiology Laboratory, University of North Texas, 1155 Union Circle, Denton, TX, USA.|Department of Biological Sciences, University of North Texas, Denton, TX, USA.
authors.forename: Adam S
authors.initials: AS
authors.lastname: Venable
authors.affiliation: Applied Physiology Laboratory, University of North Texas, 1155 Union Circle, Denton, TX, USA.|Department of Biological Sciences, University of North Texas, Denton, TX, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Henning
authors.affiliation: Applied Physiology Laboratory, University of North Texas, 1155 Union Circle, Denton, TX, USA.|Department of Biological Sciences, University of North Texas, Denton, TX, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Prado
authors.affiliation: Applied Physiology Laboratory, University of North Texas, 1155 Union Circle, Denton, TX, USA. brian.mcfarlin@unt.edu.|Department of Biological Sciences, University of North Texas, Denton, TX, USA. brian.mcfarlin@unt.edu.
authors.forename: Brian K
authors.initials: BK
authors.lastname: McFarlin
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008070:Lipopolysaccharides; D009000:Monocytes / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D059010:Single-Cell Analysis / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics
chemical_list: D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha
keywords: Cytokines; Flow cytometry; FlowRNA; FlowSight; Lipopolysaccharide; Peripheral blood mononuclear cells; QuantiGene; TNF-α; mRNA