Rat BMSC Exosome Isolation Protocol

Rat bone marrow mesenchymal stromal cells (BMSCs) were harvested and cultured with DMEM medium containing 10% FBS as previously described [33 (link)]. When the cell confluency reached 30–50%, the original medium was discarded and DMEM basic medium without FBS was added after the cells were washed with PBS three times. After 48–72 h, the cell culture supernatant was collected and underwent ultracentrifugation to collect exosomes. The detailed steps are as follows: centrifuge at 300 g for 10 min, take the supernatant, and remove the pellets to remove the cells; centrifuge at 2000 g for 10 min, take the supernatant, and discard precipitate to remove dead cells; centrifuge at 10,000 g for 30 min to remove the cell fragments in the sediment; centrifuge at 100,000 g for 70 min by ultracentrifugation; resuspend pellets at the bottom of the centrifuge tubes with a small amount of PBS. The concentration was quantified with a BCA reagent kit (#23225, Thermo Fisher Scientific, USA).