Journal
ONCOTARGET
Volume 8, Issue 25, Pages 40514-40532Publisher
IMPACT JOURNALS LLC
DOI: 10.18632/oncotarget.17121
Keywords
cell cycle; EdU; S phase; DNA replication
Categories
Funding
- Calouste Gulbenkian Foundation grant [96526]
- IMM-Lisbon fellowship [iMM/BPD/60-2016, PTDC/BEX-BCM/5899/2014]
- [SFRH/BD/45502/2008]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/45502/2008] Funding Source: FCT
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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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