Nanoparticle tracking analysis

EV samples were diluted in sterile NaCl 0.9% before nanoparticle tracking analysis (NTA). NTA was undertaken using the NanoSight NS300 (Malvern Instruments, Malvern, UK) equipped with a 405 nm laser. Ninety-second videos were recorded in five replicates per sample with optimised set parameters (the detection threshold was set to 7 and 5, respectively, for lEVs and sEVs). Temperature was automatically monitored and ranged from 20°C to 21°C. Videos were analysed when a sufficient number of valid trajectories was measured. Data capture and further analysis were performed using the NTA software version 3.1. At least three independent biological samples of each EV subtype were analysed, and the presented results correspond to the mean of the five videos taken for a given biological sample.