Isolation and Characterization of Small EVs

Small EVs were obtained from cell supernatants, as previously described with some modifications [24 (link),27 (link),69 (link)]. In brief, cells were incubated in DME medium with ultracentrifuged 5% FBS for 48 hours. The supernatants were collected and centrifuged at 300 g for 5 min and then at 2000× g for 10 min. The supernatant was then centrifuged at 10,000× g for 30 min, followed by filtration through a 0.2-μm pore filter (17597K, Sartorius, Gottingen, Lower Saxony, Germany). The collected supernatant was then subjected to preparation procedures by either ultracentrifugation at 100,000× g for 70 min as previously described [24 (link),25 (link)] or an affinity-based method using MagCapture (Fujifilm Wako Chemicals, Tokyo, Japan) [70 (link)] for small EVs isolation. Nanoparticle tracking analysis were performed using a NanoSight LM10 system (Malvern Panalytical, Westborough, MA, USA). The amount of dsDNA in EVs was determined by quantitative Real-Time PCR using these primers: human LINE1, 5′-CAAACACCGCATATTCTCACTCA-3′ (forward), and 5′-CTTCCTGTGTCCATGTGATCTCA-3′ (reverse) [23 (link),24 (link)].