title: Accurate and semi-automated analysis of bacterial association with mammalian cells.
abstract: To efficiently and accurately quantify the interactions of bacteria with mammalian cells, a reliable fluorescence microscopy assay was developed. Bacteria were engineered to become rapidly and stably fluorescent using Green Fluorescent Protein (GFP) expressed from an inducible Tet promoter. Upon application of the fluorescent bacteria onto a monolayer, extracellular bacteria could be discriminated from intracellular bacteria by antibody staining and microscopy. All bacteria could be detected by GFP expression. External bacteria stained orange, whereas internalised bacteria did not. Internalised bacteria could thus be discriminated from external bacteria by virtue of being green but not orange fluorescent. Image acquisition and counting of various fluorophore-stained entities were accomplished with a high-content screening platform. This allowed for semi-automated and accurate counting of intracellular and extracellular bacteria.
authors.affiliation: Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, The University of Dublin, Ireland.
authors.forename: C M
authors.initials: CM
authors.lastname: Murphy
authors.affiliation: Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, The University of Dublin, Ireland.
authors.forename: S
authors.initials: S
authors.lastname: Paré
authors.affiliation: School of Biology & Environmental Science, University College Dublin, Ireland.
authors.forename: G
authors.initials: G
authors.lastname: Galea
authors.affiliation: School of Biology & Environmental Science, University College Dublin, Ireland.
authors.forename: J C
authors.initials: JC
authors.lastname: Simpson
authors.affiliation: Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, The University of Dublin, Ireland. Electronic address: steve.smith@tcd.ie.
authors.forename: S G J
authors.initials: SG
authors.lastname: Smith
mesh_terms: D000818:Animals; D001419:Bacteria / Q000737:chemistry / Q000166:cytology* / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D016466:CHO Cells / Q000382:microbiology*; D002460:Cell Line; D003412:Cricetulus; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D054884:Host-Pathogen Interactions; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D011401:Promoter Regions, Genetic; D012486:Salmonella typhimurium / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins
keywords: Escherichia coli; GFP; High content analysis; Image analysis; Inducible gene expression; Invasion; Microscopy; TetR