The mitotic shake-off method revealed the remarkable variation of radiosensitivity of HeLa cells during the cell cycle: M phase shows the greatest radiosensitivity and late S phase the greatest radioresistance. This method harvests all M-phase cells with a round shape, making it impossible to further subdivide M-phase cells. Recently, the fluorescent ubiquitination-based cell cycle indicator (Fucci) was developed; this system basically causes cells in G1 to emit red fluorescence and other cells to emit green fluorescence. Because the green fluorescence rapidly disappears at late M phase, two-dimensional flow cytometry analysis can usually detect a greenhigh/redlow fraction including S-, G2- and early M-phase cells but not a transitional fraction between greenhigh/redlow and greenlow/redlow including late M-phase cells. However, combining the shake-off method concentrated the transitional fraction, which enabled us to separate early and late M-phase cells without using any drugs. Here we demonstrate for the first time that cells in early M phase are more radiosensitive than those in late M phase, implying that early M phase is the most radiosensitive sub-phase during the cell cycle.

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