Zetaview instrument

EVs were harvested according to previously described methods [34 ]. Briefly, the cell culture medium was collected and centrifuged at 2000 x g for 10 min to remove cellular debris. The supernatants were then collected and centrifuged at 10,000 x g for 30 min, and the new supernatants were collected and ultracentrifuged at 100,000 x g for 75 min. Next the deposit was obtained and resuspended with 1 ml phosphate-buffered saline (PBS) and the resuspension was ultracentrifuged again at 100,000 x g for 75 min after filtering with a 0.22 μm filter (Steritop™ Millipore, MA, USA). The new deposit (P-MSC-EVs) was resuspended with PBS and stored at −80°C. All the centrifugation steps were conducted at 4°C.
In addition, for the identification of P-MSC-EVs, a transmission electron microscope (HITACHI HT7700, Hitachi High-Technologies, Japan) was used to capture the morphology, a Zetaview instrument (Particle Metrix, Meerbusch, Germany) was used to measure the density and size, and western blotting was used to examine the expression of CD9, tumor susceptibility gene101 (TSG101), and calnexin in the cell lysis, EVs-depleted medium and P-MSC-EVs.

Exosomes were isolated from cell culture supernatants with 10% exosome‐depleted FBS (VivaCell Biosciences, China) through differential centrifugation, as described (Lötvall et al., 2014). After 48 h of culture, when cells were 90% confluent, 210 ml of the supernatant was harvested and centrifuged at 300 × g for 10 min, 2000 × g for 10 min, and then 10,000 × g for 30 min. Next, the supernatant was centrifuged at 120,000 × g for 70 min (Type 45 Ti Rotor, Beckman Coulter, USA), the pellet was resuspended in 35 ml of Phosphate buffered saline (PBS), and the sample was centrifuged again at 120,000 × g for 70 min. The supernatant was carefully removed, and the sample was resuspended in 200 μl of PBS. Exosomes isolated from human serum samples were obtained from six diagnosed PCa patients. Informed consent was obtained from all patients. Patient serum exosomes were isolated as described above and resuspended in 100 μl PBS. A BCA Protein assay kit (Pierce™, Thermo Fisher Scientific, USA) was used for exosome quantification, and exosome morphology was analysed using transmission electron microscopy (TEM) (Tecnai, USA), as previously described (Shelke et al., 2014). To examine exosome size distribution and particle concentration, nanoparticle tracking analysis (NTA) was performed using a ZetaView instrument (Particle Metrix, Germany).