Zetaview particle tracker

Serum exosomes eluted in 100 µL Buffer XE using exoRNeasy Serum/Plasma Kits were diluted in PBS to a final volume of 1 ml. The size distribution and concentration of exosomes were analysed by nanoparticle tracking analysis using ZetaView particle tracker (Particle Metrix). Ideal measurement concentrations were found by pre‐testing the ideal particle per frame value (140 ~ 200 particles/frame). The manufacturer's default software settings for exosomes were selected accordingly. Measurement data from the ZetaView were analysed using the corresponding software (ZetaView 8.02.28).

EVs were collected by density gradient ultracentrifugation as described previously.^[62] THP‐M culture medium was centrifuged at 800 × g for 5 min, followed by centrifugation at 2000 × g for 10 min to eliminate cellular debris. The supernatant was then passed through a 0.22‐µm PVDF filter (Millipore, USA) and centrifuged at 100 000 × g for 90 min at 4°C. The supernatant was discarded, and the pellets were washed with PBS and used to suspend the pellets. EVs were isolated from plasma of patients with NSCLC by use of Minute Hi‐Efficiency Exosome Precipitation Reagent (Invent Biotechnologies, USA). For removing large debris, 1‐mL plasma samples were centrifuged for 10 min at 2000 × g. Two volumes of EVs precipitation reagent were added to the supernatant, followed by 1 h of incubation. Purified EVs were obtained by centrifugation of the sample for 30 min at 10 000 × g, followed by centrifugation at 100 000 × g for 70 min. The size distribution and concentration of EVs were determined with a ZetaView particle tracker from ParticleMetrix (Germany). The shape of EVs was assessed with a transmission electron microscope (Tokyo, Japan). CD63, Alix, and TSG101 served as markers of EVs; GM130 served as a marker of cells. The concentrations of EVs were quantified with BCA protein assay kit (Beyotime Biotechnology, China).