Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis
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Pedro D. Pereira1,*, Ana Serra-Caetano1,*, Marisa Cabrita2, Evguenia Bekman1, José Braga1, José Rino1, Renè Santus3, Paulo L. Filipe1, Ana E. Sousa1 and João A. Ferreira1
1Instituto de Medicina Molecular, Faculdade Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
2Kennedy Institute of Rheumatology, University of Oxford, OX3 7FY Oxford, United Kingdom
3Muséum National d´Histoire Naturelle, Département RDDM, 75231 Paris, France
*These authors contributed equally to this work
João A. Ferreira, email: [email protected]
Keywords: cell cycle, EdU, S phase, DNA replication
Received: May 20, 2016 Accepted: April 03, 2017 Published: April 15, 2017
We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2′-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.
Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.
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