Cell labquanta sc mpl analysis software

After washing in TNE buffer, sperm samples were treated with acid-detergent solution (0.08 N HCl, 150 mM NaCl, 0.1% Triton-X 100, pH 1.2) to induce DNA denaturation. After exactly 30 s, acridine orange (AO, Sigma, St. Louis, USA) staining solution (0.6 mg AO, 0.1M citric acid, 0.2M Na₂PO₄, 1mM EDTA, 0.15 M NaCl, pH 6.0) was added to sperm suspension30 (link). Flow cytometry analysis was performed using a Beckman Coulter flow cytometer (Cell LabQuanta SC MPL, Beckman Coulter, Fullerton, CA, USA) equipped with a 488-nm argon-ion laser. For each analysis, at least 20,000 events were collected, at a rate of 200–250 events/sec. were collected and analysed using Cell LabQuanta SC MPL Analysis software (Beckman Coulter). The percentage of sperm cells with DNA fragmentation emitting strong red fluorescence (% DFI) and the percentage of incompletely condensed sperm cells with high DNA staining emitting strong green fluorescence (% HDS) were calculated. Fluorescence measurements were repeated three times using separate samples.

The analysis of sperm plasma membrane integrity (sperm viability) was assessed using propidium iodide (PI, L-7011, LIVE/DEAD®Sperm Viability Kit, Molecular Probes, ThermoFisher Scientific, Waltham, MA United States). The spermatozoa were stained in dark (4°C, 10 min) in PBS (137 mM NaCl, 2.7 mM KCl, 1.76 mM KH₂PO₄, 8.1 mM Na₂HPO₄·2H₂O, pH 7.4) staining solution with a final concentration of 1.5 × 10⁶ spermatozoa/mL and 0.48 mM PI. The flow cytometry analysis was performed using a Cell Lab Quanta ™ SC MPL flow cytometer (Beckman Coulter, Fullerton, CA, United States), equipped with a 22 mW argon laser with an excitation wavelength of 488 nm. Data were analyzed using Cell Lab Quanta SC MPL Analysis software program (Software Version 1.0, Beckman Coulter, Fullerton, CA, United States). Side scatter (SS) signals were used for the identification of spermatozoa, and PI fluorescence in SS-gated spermatozoa was detected using a 670 nm long pass (LP) filter (FL3).

Cytofluorometric evaluation of sperm suspensions was performed using a Beckman Coulter flow cytometer (Cell Lab Quanta SC MPL, Beckman Coulter, Fullerton, CA, USA) equipped with a 488-nm argon-ion laser for excitation. Samples were measured at a flow rate of 150–250 cells per second. For each analysis, at least 10,000 events were acquired. The sperm population was gated on the basis of the electronic volume (EV, parameter depending on the cell size) and side scatter signals (SS, parameter depending on cellular granules). The intensity of green (480–550 nm, for FITC) and/or red fluorescence (590–670 nm, for M540, PI) was detected using the FL1 and FL3 channels, respectively. MitoSOX Red fluorescence was detected in channel FL2 (561–550 nm). The fluorescence data were obtained at a fixed gain setting in logarithmic mode (FL1, FL2, FL3). For acquisition and data analysis, the Cell LabQuanta SC MPL Analysis software (Beckman Coulter) was used. Fluorescence reading was repeated two times with distinct samples. Binding specificity was checked with a fluorescence microscope (Olympus) (Fig. 2).