N-NPSCs and D-NPSCs were cultured in exosome-free medium for 2 days. NPSC-exo was isolated from supernatant by ultracentrifugation. The culture medium was centrifuged at 1,000 × g for 10 min at 4°C to remove dead cells and centrifuged at 10,000 × g for 30 min at 4°C to remove cell debris. Next, exosomes were centrifuged by ultracentrifugation at 100,000 × g for 70 min after filtering through 0.22-μm membrane filters. Moreover, exosomes were purified by a commercial kit using ExoJuice (WeinaBio, China) according to the instruction of the manufacturer. Briefly, exosome samples were transferred to 5-ml ultracentrifuge tubes and 500 µl of Exojuice was added to the bottom. The tube was then centrifuged at 100,000 × g for 70 min at 4°C, carefully recovered, and fractionated from the bottom. The first 200 µl of the liquid from the bottom of the tube was discarded, then the next 200 µl fraction of solution from the bottom was collected, which contained the purified exosomes.
After purification, morphology was observed by transmission electron microscopy, particle diameter and concentration were analyzed by flow nano analysis, and the exosomal markers, such as CD9, CD81, and TSG101 (Affinity, USA) were detected by Western blotting assay.
After purification, morphology was observed by transmission electron microscopy, particle diameter and concentration were analyzed by flow nano analysis, and the exosomal markers, such as CD9, CD81, and TSG101 (Affinity, USA) were detected by Western blotting assay.