Dio solution

The exo (50 µg) were incubated with the lipophilic tracer DiO solution (V22886, Thermo Fisher Scientific) at 37 °C for 20 min. The DiO-labeled exo were then added to the culture medium of recipient cells (THP-1) for 3 h of incubation at 37 °C. Later, the THP-1 cells were fixed with 4% paraformaldehyde at room temperature for 10 min and permeabilized with 0.1% Triton X-100 for 5 min. The cells were then stained with wheat germ agglutinin (WGA, W849, Thermo Fisher Scientific) conjugated to a fluorescent dye at room temperature for 30 min. The slides were mounted with Prolong™ Live Antifade Reagent (P36975, Thermo Fisher Scientific) and covered with a coverslip for observation under the Nikon eclipse 80i fluorescence microscope (DiO: excitation/emission = 480/510 nm; WGA: excitation/emission = 555/580 nm).

Conditioned medium from 2 × 10⁶ ASCs was collected and passed through a 0.8 μm vacuum filter (Millipore, USA). According to the manufacturer's instruction, the EVs were collected with exoEasy Maxi Kit (Qiagen, Germany) to a final volume of 100 μl and stored at -80°C for further analysis. EVs were identified by detecting tetraspanin protein CD81 and CD63 by western blots. The morphology of EVs was detected using transmission electron microscopy (Hitachi H7500 TEM, Japan).
EVs were incubated with DiO solution (Thermo, USA) for 20 min at 37°C. Excessive DiO was removed from Exosome Spin Columns (Thermo, USA). For immunocytochemistry, DiO-labeled EVs were added to the 1 × 10⁵ ASC culture media and incubated for 3 h at 37°C.