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

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.

High Resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

“…In order to estimate the frequency of active origin firing within TTRs, we calculated the distribution of fork speed inferred by dividing TTR sizes by fractions of S phase traversed by TTRs in human and mouse cell lines. Assuming a 10hr S phase (the maximum for the 5 cell lines profiled), the median speed of fork progression for both human and mouse was between 1.7kb/min and 1.8kb/min (with the exception of HCT116, which has a median of 2kb/min) and agrees with the range of estimated fork speed measured directly by DNA fibre methods by others ( Fig2e ) (Pereira et al, 2017; Wilson et al, 2016). Although we cannot rule out the possibility of sequential origin firing with inter-origin distances <50kb, our limit of detection, there would need to be a nearly uniform density and firing efficiency of origins along the length of each TTR to escape detection.…”

High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells

“…Assuming a 10-h S phase (the maximum for the 5 cell lines profiled), the median speed of fork progression for both human and mouse was between 1.7 and 1.8 kb/ min (with the exception of HCT116, which has a median of 2 kb/min) and agrees with the range of estimated fork speed measured directly by DNA fibre methods by others ( Fig. 2e) [32,33]. Although we cannot rule out the possibility of sequential origin firing with interorigin distances < 50 kb (our limit of detection), there would need to be a nearly uniform density and firing efficiency of origins along the length of each TTR to escape detection.…”

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency

“…Overall, our time lapse analysis shows that as mESCs transit from naive to formative pluripotency, there is a reconfiguration of the cell cycle that promotes an increased proliferation rate, which is readily detected during the first 10 h since differentiation stimulus (cells in the 2nd generation). To further validate these results, we analyzed the incorporation of the nucleoside analog EdU 23 followed by propidium iodide staining at 0, 24 and 48 h during the transition to formative pluripotency. This analysis showed a statistically significant increase in the proportion of cells in the S-phase at 48 h (Fig.…”