Exoquick tc solution

Cell culture media were centrifuged at 200 g for 10 min and then at 2000 g for 20 min at 4° C to remove debris and dead cells. Subsequently, the cell-free culture supernatant (4 mL) was incubated with 1 mL of ExoQuick-TC solution (System Biosciences, Palo Alto, CA, USA) overnight at 4° C, and exosomes were isolated by centrifugation at 1500 g for 30 min. The precipitates were resuspended in 1 mL of PBS, further purified by ultracentrifugation at 100,000 g for 70 min, and finally resuspended in 100 μL of PBS. Exosomes were detected using high-resolution transmission electron microscopy and nanosight tracking analysis (NTA; NanoSight NS300, UK). The expression of TSG101 and CD63 (specific protein markers of exosomes) were determined using western blotting analysis and the respective primary antibodies TSG101/HSP90/CD63 (rabbit anti mouse, 1:1000; Abcam, Cambridge, UK) and secondary antibody IgG (donkey anti rabbit, 1:10000; Jackson Laboratory, Bar Harbor, ME, USA). The isolated exosomes were stored at -80° C until use. For the uptake assay, exosomes labeled with PKH26 (Sigma-Aldrich, Merck KGaA, Germany) were incubated with HUVECs for 24 h and nuclei were counterstained with DAPI (blue), and the cells were then photographed.

Exosomes were isolated from the culture supernatant of cells. About 20 mL of each cellular supernatant was collected and centrifuged at 3000 × g for 15 minutes at 4°C to remove cellular debris. The supernatant was mixed with 4 mL of ExoQuick–TC solution (System Biosciences Inc, Mountain view, CA, USA) and incubated at 4°C overnight. The mixture was then centrifuged at 10,000 × g for 15 minutes at 4°C, and the precipitates were obtained to be used for subsequent experiments.