Exosome Isolation from Cancer Cell Lines

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)).