Facsaria 2 cell sorter flow cytometer

Sperm plasma membrane potential (Em) was assessed using different concentrations of DiSC₃(5) for 10 min, for proper determination of dye concentration. Unless specified, dye concentration was then of 50 nM. In addition, 2 μM of PI was added 30 s before collecting data to monitor viability. Data were recorded as individual cellular events using a BD FACSAria^TM II cell sorter flow cytometer (BD Biosciences). Forward scatter (FSC) and side scatter (SSC) fluorescence data were collected from 20,000 events per sample. Debris and cell aggregates were excluded from analysis by gating side vs. FSC cytogram. Negative stain for PI was selected for living cells and positive cells for DiSC₃(5) were detected using the filter for allophycocyanine (APC) (660/20). Normalization was performed by adding 1 μM valinomycin and 39.6 mM KCl. Data were analyzed using FACS Diva and FlowJo software (Tree Star 10.0.7r2).

The cell viability was analyzed in triplicate using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) following the steps indicated by the manufacturer. Viable cells are FITC Annexin V and propidium iodide (PI) negative and death cells are both FITC Annexin V and PI positive. Subsequently, a reading was performed on the BD FACSAriaTM II Cell Sorter flow cytometer.