2.9. Exosome extraction, identification and quantification

6 × 10⁵ normal macrophages were seeded in 10 cm dishes and cultured for 24 h. For the collection of M0 and M2 (20 ng/mL IL-4 for 1 d; with PBS washing) macrophage-derived supernatants, serum-free medium was added for another 24 h, and supernatants were collected. sh-NC and sh-Ckip-1 macrophage-derived supernatants were also collected after culturing with serum-free medium for 1 d. To extract M0 and M2 macrophage-derived exosomes (M0-EXO; M2-EXO), sh-NC and sh-Ckip-1 macrophage-derived exosomes (sh-NC-EXO; sh-Ckip-1-EXO), the above supernatants were subjected to centrifugation at 300g for 10 min; 2000 g for 10 min; 10000 g for 30 min. After 0.22 μm filtration, the supernatants were centrifugated at 120000 g for 90 min (XE-100; Beckman). The precipitation was resuspended with PBS, and again centrifugated at 120000 g for 90 min. The obtained exosomes were dissolved with 50–100 μL PBS according to the amount of precipitation.

For exosome identification, 20 μg exosomes resuspended with loading buffer were denatured at 95 °C for 10 min. Positive protein markers and negative protein marker of exosomes were detected by Western blotting. Also, the morphology of exosomes was determined by a transmission electron microscope (TEM; JEM-2100; JEOL). The sizes of exosomes were examined by dynamic light scattering (DLS) method with a nanometer particle potentiometer (Zetasizer Nano ZSP; Malvern). The concentration of exosomes dissolved in PBS was determined by the BCA kit (Beyotime) according to the manufacturer's instruction.