7 amino actinomycin d 7 aad via probe

The analyses were conducted with an EPICS XL Flow Cytometer (Coulter Electronics, IL, Italy), equipped with an argon laser at 488 nm and the following three fluorescence detectors: green (FL-1 at 525 nm), orange (FL-2 to 575 nm) and red (FL-3 at 620 nm). For each sample, 100 000 events were measured at low flow velocities and analyzed using Sistem II™, version 3.0.
To obtain the absolute leukocytes counts, 100 μl of each liquefied semen sample was incubated with a mixture containing Syto-16 green fluorescent nucleic acid stain to identify the spermatozoa and exclude debris (the final concentration was 200 nmol l⁻¹, Molecular Probes, Eugene, Oregon, USA), 7-amino-actinomycin D (7-AAD Via-Probe, BD Pharmingen, San Diego, CA, USA) to assess viability, anti-CD45-APC (pan-leukocyte antigen) to recognize white blood cells and anti-CD16-PE for PMN recognition. The addition of 100 μl of Flow-Count™ Fluorospheres (Beckmann-Coulter, Fullerton, CA, USA) at 1034 beads per ml allowed for the determination of the absolute leukocyte count by flow cytometry. After incubation in the dark for 20 min at room temperature, 1 ml of PBS was added and the sample was analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA). For each test, 100 000 events were acquired.