Beckman optima l 90k

Fresh grapefruits and tomatoes were used as PEV sources. These fruits were purchased from a local market in Gatchina, Russia. The juices were extracted using a household juicer (Moulinex Y36-Vitafruit, Alençon, France), then each juice was filtered once using a PEV isolation technique, which was performed according to protocols in other studies [7 (link),15 (link),16 (link)].
Briefly, the filtered juices of grapefruits and tomatoes were sequentially centrifuged using an Avanti J30-I centrifuge (JA-10 rotor, Beckman Coulter, Brea, CA, USA) at 1500× g for 30 min, 3500× g for 20 min, 10,000× g for 60 min, 16,000× g for 60 min, and 10,000× g overnight to remove large particles and cellular debris. The supernatant was subjected to ultracentrifugation using a Beckman Optima L-90K ultracentrifuge (Ti 45 rotor, Beckman Coulter, Brea, CA, USA) at 150,000× g for 2 h. Then, the supernatant was removed, and the pellet was carefully resuspended in 2 mL of 1× PBS by gentle swaying overnight. The volume was adjusted to 10 mL with 1× PBS and ultracentrifuged at 150,000× g for 2 h (Ti 70 rotor, Beckman Coulter, Brea, CA, USA). The resulting pellet was resuspended with 1 mL of 1× PBS for at least 1 h at 4 °C. Final samples of grapefruit exosome-like vesicles (GEVs) and tomato exosome-like vesicles (TEVs) were aliquoted, snap frozen in liquid nitrogen, and stored at −80°C until analysis.

The exosome-producing medium was prepared to remove residual exosomes from FBS as referred [35 ] with modification. Generally, 50% (v/v) FBS in DMEM medium was centrifuged at 2,000 × g for 10 min, and then centrifuged at 100,000 × g (Beckman Optima L90-K with 90Ti rotor; Beckman Coulter Taiwan Inc., Taipei, Taiwan) for 16 hr at 4° C. The supernatant were collected and diluted into 10% (v/v) FBS by serum-free DMEM, and were sterile through 0.22 μm filter. For production of hepatoma-derived exosomes, 1 × 10⁶ Hep3B or Huh7 cells were plated in culture medium overnight, and were replaced into exosome-producing medium for successive culture for 2 days. Exosome-containing medium (100 mL) were collected and exosomes were isolated by Total Exosome Isolation kit (Life Technologies, Grand Island, NY, USA) according to suggested protocol. The pellets containing secreted exosomes were further washed by DEPC-treated PBS, centrifuged at 100,000 × g for 60 min (Beckman Optima MAX-E with TLA-120.2 rotor), and repeated twice to remove residual serum protein. The protein content in the exosome solution was determined by protein assay reagent (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).

To avoid contamination, FBS was filtered with a 0.22 µm filter (Merck, Burlington, MA, USA), followed by ultracentrifugation at 120,000× g for 18 h in a fixed-angle rotor 70ti (Beckman Optima L-90K, Beckman Coulter, Brea, CA, USA). FBS-derived EVs were pelleted at the bottom of the tube, and the supernatant was collected as EV-depleted FBS. For EVs isolation from the culture medium, B6-DPCs were cultured in a 100 mm dish up to 70–80% confluence. The cells were washed three times with phosphate-buffered saline (PBS) and then cultured in DMEM/10% FBS (EV-depleted) for 48 h. Serial centrifugations were conducted at 4 °C from cell pellet removal to EV purification. First, a 10 min 300× g centrifugation was used to eliminate the cell pellet, then centrifugation at 2000× g for 20 min was employed to eliminate the dead cells. To eliminate cell debris and large particles, centrifugation was performed at 10,000× g for 30 min. Thereafter, the supernatant was collected and non-gradient ultracentrifugation was performed at 100,000× g for 70 min. For EV purification and elimination of protein contamination, supernatant was gently removed. The pellet was resuspended in cold PBS, and ultracentrifugation at 100,000× g was performed for 70 min. After resuspension, the EVs were preserved at −80 °C.