Exosome Isolation from Cell Culture

Exosomes were isolated from cell culture conditioned supernatants using the protocol published by Lobb et al. [50 (link)]. Conditioned culture media were harvested from adipocytes and centrifuged at 300× g at 4 °C for 10 min to remove detached cells. Then, supernatants were filtered through 0.22 µm filters (Merck Millipore, Darmstadt, Germany) to remove contaminating apoptotic bodies, microvesicles and cell debris. Clarified conditioned culture media were then centrifuged in a Sorvall^TM MTX 150 micro-ultracentrifuge (Thermo Scientific, Waltham, MA, USA) at 100,000× g at 4 °C for 90 min to pellet exosomes. The supernatants were carefully removed, and pellets containing exosomes were resuspended in 1 mL of ice-cold PBS. A second round of ultracentrifugation under the same condition was carried out, and the resulting exosome pellets resuspended in 200 µL of PBS.