Exosome Isolation from Seminal Plasma

Exosomes were purified from the entire cell- and debris-free seminal plasma (ranging from 0.9 to 4.0 ml in our donors) by ultracentrifugation over a sucrose cushion, as previously described (Vojtech et al., 2014 (link)). Up to 2.5 ml of supernatant was added to ultracentrifuge tubes and under-layered with 300 μl of a 20 mM Tris/30% sucrose/deuterium oxide (D₂O) cushion (pH 7.4) (Sigma, St. Louis, MO, USA). Samples were ultra-centrifuged at 100 000 ×g for 90 min at 4°C in an SW 50 swinging bucket rotor (Beckman, Brea, CA, USA). The upper layer was collected and ultracentrifuged again at 100 000 ×g for 14 h at 4°C over a 20 mM Tris/25% sucrose/D₂O cushion (pH 7.4). The upper layer of exosome-depleted seminal plasma was stored at −80°C. The 30% and 25% sucrose cushions containing the exosome fraction were pooled and brought to 15 ml with Dulbecco’s PBS (DPBS, GIBCO Thermo Fisher Scientific, Bothell, WA, USA). The exosomes were washed by centrifuging at 2400 ×g at 20°C through an Amicon Ultracel 100 kDa cellulose centrifugal filter (Millipore Sigma, St. Louis, MO, USA) with 10 ml of PBS and concentrated to a final volume of 425 μl–3.2 ml. Exosomes were stored at −80°C.