Quantifying Oxidative Stress via Luminol Assay

To determine the extent of oxidative stress, the quantities of ROS produced by the control and experimental samples were quantified by the chemiluminescent assay based on the ability of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma-Aldrich, St. Louis, MO, USA) to interact with reactive intermediates. The samples (100 μL) were pipetted onto clear 96-well plates, carrying blank (100 μL PBS), negative control (100 μL PBS) and positive control (100 μL PBS, 12.5 μL 30% hydrogen peroxide (H₂O₂; Sigma-Aldrich, St. Louis, MO, USA)). Subsequently, the samples and controls were treated with 2.5 μL of luminol working solution and the resulting light signal was monitored in 15 consecutive one-minute-long cycles using the Glomax Multi+ spectro-fluoro-luminometer. The results are expressed as relative light units (RLU)/s/10⁶ spermatozoa [31 (link)].