Exosome Isolation and Characterization Protocol

Exosome isolation, characterization (by TEM and western blotting) and storage were performed as previously reported by us and others [27 (link),29 (link)]. In brief, starting from passage three, the MSCs culture medium was removed when the cells reached 80% confluency, and the serum-free medium was replenished and left for 24 h; it was collected and now called conditioned medium (CM). The collected CM was filtered through a 0.22 μm filter (to remove debris and dead cells) and was centrifuged at 10,000 g for 30 min at 4 °C and the supernatant was collected. The supernatant was then subjected to ultracentrifugation at 100,000 g for 60 min at 4 °C to pellet the exosomes. The pellet was washed twice with ice-cold PBS, and ultracentrifugation was performed for each wash to re-pellet the isolated exosomes. To quantify the amount of isolated exosomes, the protein content was measured by standard Bradford assay, and then the isolated exosomes were adjusted to 100 µg protein in 200 µL aliquots and stored at −80 °C. The characterization of isolated exosomes was performed using the detection of exosomes’ markers, CD63 (sc-5275, Santa Cruz Biotechnology, USA) and CD81 (sc-166029, Santa Cruz Biotechnology, Dallas, Texas, USA), by western blotting and by transmission electron microscopic (TEM) detection of their shape and size.