Staphylococcus aureus strain ATCC 1718 was inoculated at 0.5% in 400 mL of Brain Heart Infusion broth for 8 h under aerobic conditions. The supernatant was collected by centrifugation twice at 5,000 x g, 4°C for 20 min, filtrated through a 0.45 µm filter to remove all remaining bacterial cells. S. aureus-derived extracellular vesicles (SaEVs) in culture supernatant were precipitated by ultracentrifugation at 100,000 x g, 4°C for 90 min using a Himac CP80WX Preparative Ultracentrifuge (HITACHI, Tokyo, Japan). After washing with ice-cold PBS, the pellet was suspended and applied to discontinuous iodixanol gradient ultracentrifugation. Layers of 40%, 20%, 10%, and 5% iodixanol in 0.25 M sucrose/10 mM Tris, pH 7.5 were prepared using OptiPrep™ [60% (w/v) iodixanol; Sigma Aldrich, St. Louis, MO]. After centrifugation at 100,000 x g, 4°C for 16 h, the substances in all six fractions (Fr1-Fr6) were collected, diluted in PBS, and precipitated by ultracentrifugation at 100,000 × g, 4°C for 2 h. The pellets were then washed and resuspended in an appropriate volume of ice-cold PBS.