Calculation of cell cycle length with EdU-BrdU double labeling

Cell cycle length analysis essentially followed a previously described method (23). Briefly, the pregnant female mice were injected intraperitoneally with EdU at a dose of 100 μg/g body weight. Then, the same dose of BrdU was injected at 1.5 hours later. Mice were euthanized at 2 hours after the first injection. Embryos were fixed, frozen-sectioned, and processed for detection of incorporated EdU using the Click-iT Imaging Kit (Invitrogen) and BrdU by anti-BrdU antibodies (MoBu-1, Abcam, ab8039). The section was costained with 4′,6-diamidino-2-phenylindole (DAPI). The cell cycle lengths (Tc) were calculated by the following equation, which is based on the assumptions that all cells in the ventricular zone are proliferating and that the length ratio of each cell cycle is equal to the ratio of the number of cells

Tc (h) = P cells × Ti (time between injections = 1.5 hours)/L cells

P cells represent the total number of proliferating cells in the ventricular zone, whereas L cells (leaving fraction) are the number of cells labeled with EdU, but not BrdU.