Extracellular Vesicle Isolation from Seminal Plasma

Immediately after CASA analysis, semen samples (10 mL) were centrifuged at 800× g for 20 min at room temperature to collect spermatozoa. Corresponding supernatants were subjected to additional centrifugation (2000× g for 20 min) at 4 °C. The resulting supernatants (seminal plasma), devoid of cell debris and residual sperm cells, were collected and stored at −80 °C until EVs isolation. Frozen-thawed seminal plasma was centrifuged at 16,000× g for one hour at 4 °C. The resulting supernatants were ultracentrifuged (120,000× g for 70 min) at 4 °C. Visible EV pellets were rinsed twice with a high-purified cold PBS (5 mL) by ultracentrifugation (120,000× g for 70 min) at 4 °C. Cleaned EV pellets were resuspended in aliquots of 100 µL cold PBS and stored at −80 °C for further analysis.