Establishment of a method for classifying green phase into early/mid/late S and G2 phases.

(A) Experimental procedure for classification of green phase. Time-lapse imaging was performed for cells flash-labeled with EdU immediately after 5 Gy irradiation, followed by EdU staining. Time is shown as hours:minutes. (B) Classification of green-phase cells into early/mid/late S and G2 phases, according to the method described in A. For simplicity, a subset of cells in each phase is presented. To adjust the fluorescence intensities among different fields, background fluorescence intensity of each image was normalized to 1. (C) Comparison of green fluorescence intensities among sub-groups. Data are represented as box-and-whisker plots, as described in Fig 3E, from 60 cells in early S, 139 cells in mid S, 101 cells in late S, and 127 cells in G2 phase. *, p < 0.05; **, p < 0.01 by Mann–Whitney U test. (D) Validation of the methodology, using GP1 and GP2 from Fig 3D. Cell numbers in each sub-phase within GP1 (a) and GP2 (b) were determined. GP1 and GP2 consisted of 138 and 289 cells, respectively.