Nanoparticle Tracking Analysis (NTA)

Suspension containing EVs from all four groups were analyzed with the Nano-Sight NS300 Instrument (Malvern, Worcestershire, United Kingdom). Samples were diluted with 0.2 μm filtered 1× PBS in order to obtain an optimal range of 20–150 particles/frame. For each sample, 5 videos of 60″ duration were recorded and data were processed using NanoSight NTA Software 3.2. Post-acquisition settings were kept constant between samples. Data obtained were: particle concentration (particles/ml), average size (nm), and particle size distribution (D10, D50, D90: particle size values indicating that, respectively, 10, 50, and 90% of the distribution is below this value). Raw concentration data (particles/ml) obtained from NTA were normalized to obtain EVs concentrations in the human plasma sample.