Fig. 2: Validation of the sorting method in HeLa cells.
Two different types of analysis were performed in HeLa cells to validate S-subphases classification on the basis of total EdU and DNA content. a, b Schematic comparison between (a) our sorting method based on the analysis of EdU pixels in a single optical section and (b) a standard method based on the evaluation of total EdU and DNA content from acquisition of multiple optical sections in 2 channels. For this comparison we used 5 optical sections. c Scatter-plot of replication foci pixel density (ρRF) versus intensity (IRF) for a population of HeLa cells, as determined from a single optical section. The gray dashed lines indicate the thresholds for separation into the 4 groups: Early S-subphase (black), Middle S-subphase (red), Late S-subphase (green), and G1/G2 phases (blue). d Scatter plot of Total EdU content vs total DNA content obtained by the z-stack analysis, for the same cells classified in (c). e Representative confocal images of EdU replication foci in HeLa cells extracted from each group. Nuclear DNA is counterstained with DAPI, using a long incubation time (2 h). Scale bar 10 µm.
