Exosome Isolation and Characterization

Exosomes were collected by density gradient ultracentrifugation according to previously published protocol^{46 (link)}. In brief, the polarized macrophages were incubated for 48 h in complete PRMI1640 medium with 10% FBS that was previously depleted of contaminating vesicles by overnight centrifugation at 100,000×g. The conditioned medium was collected and centrifuged at 800×g for 10 min, followed by a centrifugation step of 3000×g for 30 min to remove cell debris. Next, the supernatant was filtered using a 0.22-µm filter (Millipore). The exosomes were pelleted by ultracentrifugation at 100,000×g for 90 min, washed in PBS, pelleted again and re-suspended in PBS. Measurement of the exosome particle number was performed using a CD63 ExoELISA Complete Kit (System Biosciences, USA) following the manufacturers’ instructions. For Nano-LC–MS/MS analysis, exosome pellets were also isolated using ExoQuick-TC TM (System Bioscience) according to the manufacturer’s protocol. For exosome uptake experiments, exosome preparations were labeled with PKH67 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer’s instructions, followed by washing through Exosome Spin Columns (MW3000) (Invitrogen, USA) to remove excess dye. Next, exosomes were incubated with GC cells, which were examined under a confocal microscope or analyzed using flow cytometry at the indicated time points.