Fig. 1

Flow cytometric out-gating of ROS sperm particles based on scatter properties and histogram after Triple-Staining. Sperm cells were selected using discontinuous density gradients method and stained with SYTOX Red, MitoSOX Red and DCFH-DA. A FSC/SSC dot plot obtained from a semen sample. A region (P1) is established to exclude debris. B SYTOX Red fluorescence histogram obtained within P1 region. The B1-L and B1-R peaks represent viable and nonviable sperm, respectively. C MitoSOX Red fluorescence distribution of viable sperm, were derived by gating on the B1-L population in panels B. The B2-R region represents ROS positive living sperm. D DCFH-DA fluorescence histogram obtained within B1-L region. The B3-R populations contains hydrogen peroxide positive live sperm events