Figure 1

Flow cytometric gating strategy for specific identification of viable, dead and apoptotic sperm, and leukocytes. Plot A shows semen population gated on the basis of scattering measurements (forward-angle scatter versus side-angle scatter); Gate 1 was made to identify the semen cell (red) and exclude the debris (black). Plot B shows the gate G4, made on Syto 16 versus SSC parameters to identify the nucleated cells stained with Syto 16. Two gates G2 (plot A) and G3 (plot B), in green, were set to accurately identify bead population. Plot C represents the events gated in G4 that were analyzed in FL4 versus SSC to identify the CD45 positive cells (leukocytes) (R5) and the CD45 negative cells (sperm) (R4). Plot D shows the events gated in R4 (sperm) that was analyzed in another cytogram, Syto 16 versus 7-AAD, to identify viable (R8) apoptotic (R7) and dead sperm (R6).