Isolation and characterization of U251 cell-derived exosomes

U251 cells were maintained in DMEM/F12 supplemented with 10% exosome-depleted FBS (EXO-FBS-50A-1, System Bioscience, Mountain View, USA) for 48 h (80–90% confluence), and the culture medium was collected and centrifuged at 800×g for 5 min and 1500×g for 15 min to remove supernumerary cells. Next, the supernatants were filtered using a Steriflip (0.22 μm, Millex-GP; Millipore, Burlington, MA, USA), and the filtrates were concentrated in a 10-kDa ultracentrifuge tube (Amicon Ultra 15; Millipore) at 4000×g for 30 min. U251 cell-derived exosomes were subsequently isolated using ExoQuick-TC™ (System Bioscience, Mountain View, CA, USA) according to the manufacturer’s directions. The mixture was refrigerated overnight at 4 °C and centrifuged at 1500×g for 30 min, and the supernatants were aspirated. The exosome-containing pellets were suspended in phosphate-buffered saline (PBS) and used immediately or stored at − 80 °C. The protein density of exosomes was measured with a BCA protein micro-assay (CWBIO, Shanghai, China). The size of exosomes was measured using a Zetasizer Nano series-Nano-ZS (Malvern Instruments, Worcestershire, UK) according to the manufacturer’s directions. The exosome markers HSP70, Tsg101, and CD9 were detected by Western blotting, and the surface markers CD63 and CD81 were detected by flow cytometry (Accuri C6; BD Biosciences, MD, USA).