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.
Highlights
The rate at which mammalian cells entry and progress through the different stages of their cell cycle is subject to strict regulatory mechanisms to avoid abnormal cell growth and division that may pose a threat to structure and function at the tissue level [1]
HCT-116 cells were synchronized at the G1/S transition by a double thymidine block and exposed to a range of EdU concentrations (5, 10, 20 and 30 μM) for a full S phase
This provides values on the proportion of cells found at each phase (G1, S and G2/M) which directly correspond to relative durations in reference to the length of a full cell cycle
Summary
The rate at which mammalian cells entry and progress through the different stages of their cell cycle is subject to strict regulatory mechanisms to avoid abnormal cell growth and division that may pose a threat to structure and function at the tissue level [1]. By identifying changes in proliferation rates in response to treatment important contributes can be made to the development of anti-cancer therapeutic agents targeting specific steps of the cell cycle and the tailoring of treatment strategies for oncologic patients. Determining cell cycle kinetics for distinct cell cycle stages is an important step for characterization of cancer cell lines [3]. Kinetics of S phase, in particular, can provide important information on control mechanisms and shifts in DNA replication. During early embryogenesis changes in S phase duration are frequent and reflect a progressive slowing down of firing rates of replication origins [4], while neuronal progenitor cells seem to shorten their S phase as they switch transcription factors on the path to neuron differentiation [5]
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