Dual laser facs aria 2

MMP in HFS and LFS was measured using rhodamine 123 (Rh123; Sigma-Aldrich, St. Louis, MO, USA). Briefly, spermatozoa were mixed with 1 μM Rh123 diluted in PBS and the concentration of spermatozoa were adjusted to 5 × 10⁶ cells/mL, and then incubated at 37 °C for 15 min. Fluorescence intensity of Rh123 was measured by flow cytometry (Dual-Laser FACS Aria II, BD Biosciences, San Jose, CA, USA) with 488 nm excitation and 525 nm emission wavelengths and analyzed. For each of the three independent replicate experiments, three samples were used.

Mitochondrial integrity was measured by rhodhamine 123 as described previously^{59 (link)}. Briefly, spermatozoa was washed with PBS and adjusted to 5 × 10⁶/mL concentration. Next, rhodhamine 123 (Rh123) was added to spermatozoa and incubated for 20 min at the room temperature in the dark. After incubation, spermatozoa were centrifuged at 100 g for 3 min. The sperm pellets were added to 1 mL of PBS and analysed with a flow cytometer. Flow cytometric analyses were performed using the Dual-Laser FACS Aria II (BD Biosciences, San Jose, CA, USA). The fluorescence intensities of Rh123 and PI of 10,000 events were recorded for each treatment. Spermatozoa with normal mitochondrial integrity, which is expressed by positive signal for Rh123 and negative signal for PI was calculated^{59 (link),60 (link)}.