Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis

// Pedro D. Pereira 1, * , Ana Serra-Caetano 1, * , Marisa Cabrita 2 , Evguenia Bekman 1 , Jose Braga 1 , Jose Rino 1 , Rene Santus 3 , Paulo L. Filipe 1 , Ana E. Sousa 1 and Joao A. Ferreira 1 1 Instituto de Medicina Molecular, Faculdade Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal 2 Kennedy Institute of Rheumatology, University of Oxford, OX3 7FY Oxford, United Kingdom 3 Museum National d´Histoire Naturelle, Departement RDDM, 75231 Paris, France * These authors contributed equally to this work Correspondence to: Joao A. Ferreira, email: hjoao@fm.ul.pt Keywords: cell cycle, EdU, S phase, DNA replication Received: May 20, 2016      Accepted: April 03, 2017      Published: April 15, 2017 ABSTRACT 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.