Exosome Isolation from Conditioned Media

Approximately 5 × 10⁶ cells were seeded in cell culture dishes. To make the data more accurate, the same number of cells were seeded in different comparison groups each time. Then, cells were cultured in normal medium until 80% confluent, followed by 3 PBS washes and 18 mL of serum-free medium culturing for an additional 40 h. Next, the serum-free conditioned medium was harvested and centrifuged sequentially at 300 × g and 2000 × g for 10 min to remove cells and cell debris, respectively. Small cell debris and larger microvesicles were removed by 10,000 × g centrifugation for 30 min (Avanti J-E, Beckman, USA). Finally, the exosome-rich pellet was collected after ultracentrifugation at 167,000 × g for 70 min (XPN-100, Beckman, USA). The exosome-rich pellet was washed by PBS and ultracentrifuged at 167,000 × g for 70 min again [42 (link), 43 (link)]. Exosomes were aliquoted for the following experiments.