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.
Highlights
Analysis of cell proliferation is essential for studies of cellular function, effects of drugs, various biological factors and treatments
Cultures were synchronized by aphidicolin (APH) blocking cells at G1/S phase, stained with 1 μM HXT for 30 min, exposed to BrdU for 4 h and analyzed by fluorescence lifetime imaging microscopy (FLIM)-time-correlated single photon counting (TCSPC) microscopy under 405 nm excitation, which was non-damaging to the cells
The new FLIM method based on HXT quenching by incorporated BrdU allows monitoring of cell proliferation in live cultures
Summary
Analysis of cell proliferation is essential for studies of cellular function, effects of drugs, various biological factors and treatments. Classical methods of analysis of cell proliferation are based on incorporation of thymidine analogues during DNA replication and/ or labeling with a suitable tracers such as 3H-thymidine, fluorescent antibody or dye reacting with 5-bromo-2’-deoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine, respectively [1,2,3]. Fluorescence-based microscopy and flow cytometry platforms have replaced the unsafe autoradiography [4, 5], but they still remain tedious, mostly end-point, suffer from antibody variability, the need of epitope unmasking, limited in-depth staining and toxicity of click-reaction products. Hoechst are a family of cell-permeable bis-benzimide dyes, which bind to the minor groove of double-stranded (ds) DNA with strong enhancement of their blue fluorescence and bright. The use of transiently or stably expressed genetically encoded fluorescent proteins fused with cell cycle markers is complex, can influence cell cycle, and have limited use with primary cells and complex 3D models [6, 7].
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