title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.
abstract: Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.
authors.affiliation: School of Biochemistry and Cell Biology, ABCRF, University College Cork, College Road, Cork, Ireland.
authors.forename: Irina A
authors.initials: IA
authors.lastname: Okkelman
authors.affiliation: School of Biochemistry and Cell Biology, ABCRF, University College Cork, College Road, Cork, Ireland.
authors.forename: Ruslan I
authors.identifier: http://orcid.org/0000-0002-0347-8718
authors.initials: RI
authors.lastname: Dmitriev
authors.affiliation: Department of Anatomy and Neuroscience, University College Cork, Western Road, Cork, Ireland.
authors.forename: Tara
authors.initials: T
authors.lastname: Foley
authors.affiliation: School of Biochemistry and Cell Biology, ABCRF, University College Cork, College Road, Cork, Ireland.
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D005455:Fluorescent Antibody Technique; D045325:HCT116 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008962:Models, Theoretical; D061848:Optical Imaging / Q000295:instrumentation*; D016196:S Phase; D018874:Spheroids, Cellular / Q000166:cytology