Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

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Papkovsky, Dmitri B.
Okkelman, Irina A.
Dmitriev, Ruslan I.
Foley, Tara
Papkovsky, Dmitri B.
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Public Library of Science
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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.
Organoids , Cell cycle and cell division , Cell staining , Fluorescence imaging , Synthesis phase , Gastrointestinal tract , Cell proliferation , Fluorescence microscopy
Okkelman, I. A., Dmitriev, R. I., Foley, T. and Papkovsky, D. B. (2016) 'Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase', PLOS ONE, 11(12), pp. e0167385. doi:10.1371/journal.pone.0167385
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