Sorvall wx ultracentrifuge series

EVs were isolated from the culture medium of LPS-treated HMDMs according to an ultracentrifugation-based method described previously [44 (link)]. In brief, dead cells and cell debris in the culture medium were removed by successive centrifugation at 300 g for 10 min, 2,000 g for 10 min, and 10,000 g for 30 min. The supernatant was then ultracentrifuged using Sorvall WX+ Ultracentrifuge Series (Thermo Fisher Scientific) at 110,000 g for 2 h. The pellet was washed with PBS and ultracentrifuged again at 110,000 g for 2 h to eliminate contaminant proteins. The final pellet was collected as the EV fraction. The data regarding EV isolation and characterization are available in EV-TRACK database (EV-TRACK ID: EV190062) [85 (link)]. To confirm the presence of EV-RNAs, the isolated EVs were incubated with PureLink RNase A (200 ng/μl, Thermo Fisher Scientific) with or without 0.1% Triton X-100 at 37°C for 30 min.

After 5 days of cell cultures, supernatants were collected, and exosomes isolated as previously described⁴⁸ (link). Briefly, after centrifugation of cells at 300 g for 5 min, supernatants were centrifuged at 1200 g for 15 min followed by 12,000 g for 30 min. Supernatants were then filtered using a 0.22-µm filter (Millipore Corp., Bedford, MA, USA) and centrifuged at 110,000 g for 1 h in a Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific, Waltham, MA, USA) to pellet exosomes. After one wash in a large volume of phosphate-buffered saline (PBS), exosomes were resuspended in appropriate buffers for subsequent experimental analysis.

Fruit juices were centrifuged at 500× g for 10 min at 10 °C; the supernatants were filtered with 100 µm filters and serially centrifugated at 2000× g for 20 min at 10 °C to eliminate cell debris and then at 15,000× g for 30 min at 10 °C to eliminate the fraction enriched in microvesicles. The supernatants were subsequently ultracentrifuged in a Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific, Waltham, MA, USA) at 110,000× g for 1 h 30 min at 10 °C to collect the Exocomplex^®. The pellet was resuspended in ultra-filtered and sterilized water (or in PBS) for downstream analyses and preserved at +4 °C.

To obtain plasma from blood samples, EDTA-treated blood from PCa patients and CTR were centrifuged at 400 x g for 20 min. Plasma was then collected and stored at −80°C until analysis. Upon thawing, 1 mL of plasma underwent the centrifugal procedure as previously described (6 (link), 41 (link)) in order to eliminate cell debris, organelles and microvesicles, and pellet exosomes. In the last step, plasma samples were centrifuged for 1 h 30 min at 110,000 x g using a Fiberlite™ F50L-24 x 1.5 Fixed-Angle Rotor, K-Factor: 33 (Thermo Fisher Scientific, Waltham, MA, USA) in the Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific), to obtain the exosomal pellet, which was then washed in PBS and resuspended in the appropriate buffer for subsequent analyzes. In particular, the exosomal pellet was resuspended in PBS for Nanoparticle Tracking Analysis and Flow Cytometry Analysis, and in CHAPS buffer 1x for western blot analysis.

PC3 and DU145 cells were cultured with cell culture medium with a pH between 6.5 and 7.5 for two weeks, and then 1.5 – 2.0×10⁶ cells were incubated in 75 cm² flasks until they reached approximately 60–70% confluence. Subsequently, the cells were further incubated with exosome-free medium (Gibco) for 24 h. Then cell culture media was collected and centrifugated at 300× g for 5 min. Following were supernatants centrifuged at 1200× g for 15 min, followed by 12,000× g for 30 min. In the end, supernatants were centrifuged at 110,000× g for 1 h in a Sorvall WX Ultracentrifuge Series (ThermoFisher Scientific, Waltham, MA, USA) in order to pellet exosomes. After one wash in a large volume of phosphate-buffered saline (PBS), and centrifuged at 110,000× g for another 1 h. At last, exosomes were resuspended in PBS (50 µL) for further analysis (27 (link)).

To obtain plasma from blood samples, EDTA-treated blood from PCa patients, BPH patients, and CTR were centrifuged at 400× g for 20 min. Plasma was then collected and stored at −80 °C until analysis. Upon thawing, 1 mL of plasma underwent the centrifugal procedure as previously described [18 (link),26 (link)] in order to pellet exosomes. Plasma samples were centrifuged for 1 h 30 min at 110,000× g using a Fiberlite™ F50L-24 × 1.5 Fixed-Angle Rotor, K-Factor: 33 (Thermo Fisher Scientific, Waltham, MA, USA) in the Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific).

Fruit juices were centrifuged at 500× g × 10 min; the supernatants were filtered with 100 µm filters and serially centrifugated at 2000× g for 20 min to eliminate cell debris and then at 15,000× g for 30 min to eliminate the fraction enriched in microvesicles. The supernatants were subsequently ultracentrifuged in a Sorvall WX Ultracentrifuge Series (Thermo Fisher Scientific) at 110,000× g for 1 h 30 min to collect the nanovesicles. The pellet was resuspended in an appropriate buffer for downstream analyses.