Exosomes were prepared by the standard ultracentrifugation method according to a previous report [
14], and this method was performed as we reported previously [
15]. In brief, A549 and HCT116 cells (3 × 10
6 cells) were seeded in a 100-mm dish in culture medium with 10% FBS. After 24 h, culture medium was changed to culture medium without FBS and incubated. COLO205 cells (6 × 10
6 cells) were seeded in a 100-mm dish in culture medium without 10% FBS and incubated. The culture media were collected after 72 h incubation and the exosomes were isolated by the following three methods: ultracentrifugation, ExoQuick-TC® (System Biosciences Inc., Palo Alto, CA, USA) and Total Exosome Isolation® (Thermo Fisher Scientific Inc., Waltham, MA, USA). The ultracentrifugation method was performed as follows. The collected medium was centrifuged at 2000
g for 30 min, and then at 10,000
g for 30 min to remove cell debris. The supernatant was centrifuged at 100,000
g for 70 min to purify exosomes. The pellet was washed with PBS and ultracentrifuged at 100,000
g for 70 min again. The pellet was resuspended with PBS and stored until use. Exosome isolation using ExoQuick-TC and Total Exosome Isolation was performed according to the manufacturer’s instructions. In brief, the collected medium was centrifuged at 2000
g for 30 min and supernatant was collected. One-fifth of ExoQuick-TC Exosome Precipitation Solution or half of Total Exosome Isolation were added to the supernatant and their suspension was incubated overnight at 4 °C. The suspension was centrifuged at 1500
g for 30 min for ExoQuick-TC or at 10,000
g for 60 min for Total Exosome Isolation. The pellet was resuspended with PBS. Exosome protein content was qualified using the BCA protein assay kit (Thermo Fisher Scientific) before further experiments.