Beckman optima max e

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).