Measuring Sperm Oxidative Stress

ROS content (mainly superoxide anion) was measured by employing the CellROX™ Deep Red probe. This allowed for the identification of spermatozoa with high superoxide anion content (sperm stained by CellROX™), as previously described by Riesco et al. [33 (link)]. Briefly, the sperm samples were diluted in PBS to obtain a total of 2 × 10⁶ spermatozoa per sample. The sperm samples were washed and centrifuged at 500× g for 10 min at room temperature. Then, cells were incubated at room temperature in the dark for 30 min with aliquots of CellROX™ (5 μM final concentration, Thermo Fisher, Madrid, Spain). To stop cell staining and avoid an overstaining effect, an additional wash was performed. Then, flow cytometry analysis was immediately conducted, with the pellet resuspended in 1 mL PBS.
Flow cytometry acquisition was performed in a flow cytometer (MACSQuant Analyser 10, Miltenyi Biotech, Madrid, Spain) controlled with MACS Quantify 2.13 software (Miltenyi Biotech, Madrid, Spain). Per sample, a total of 50,000 events and at least 20,000 sperm cells, at a flow rate of 200–300 cells per second, were acquired. FlowJo v.10.2 (Ashland, Wilmington, DE, USA) was used to analyze the data.