Exoquick tc ultra

Exosomes were isolated from culture media, collected 48 h after cell starvation using sequential centrifugation or by ExoQuick-TC-ULTRA (System Biosciences, Palo Alto, CA, USA). The ultracentrifugation was performed as previously described [30 (link)] and included centrifugation at 3000× g for 15 min, followed by 10,000× g for 30 min and 110,000 for 3 h. The resulting exosome pellet was washed in phosphate-buffered saline (PBS) and centrifuged again at 110,000× g for 70 min. The final pellet was resuspended in PBS and used in further experiments. Exosome isolation by ExoQuick -TC ULTRA was performed following manufacturer’s instruction and included removal of cell debris at 3000× g for 15 min and precipitation with the ExoQuick-TC reagent overnight at 4 °C. After incubation, the solution was centrifuged at 3000× g for 10 min and exosomes were added to ExoQuick ULTRA columns, which were centrifuged at 1000× g for 30 s. The exosomes were then collected and diluted in PBS. The protein concentration of exosomes was determined by a Bradford assay (BioRad, Hercules, CA, USA).