Zetaview software 8

Vesicle concentrations and sizes were measured by NTA using a PMX-220 ZetaView camera (Particle Metrix) with samples diluted in particle-free PBS (pfPBS), which PBS clarified immediately prior to use by 100 nm pore size filtration (to minimize the presence of nanometer scale phosphate crystals). NTA measurements were carried out in triplicate at ambient temperature with fixed camera settings. Fluorescent nanoparticle tracking analysis (flNTA) was performed by incubating Alexa Fluor 488-conjugates of monoclonal anti-CD63 antibodies with exosome suspensions (2 h, RT, in absence of light), diluting them in pfPBS, and then interrogating them using a PMX-220 ZetaView camera (Particle Metrix). Samples were visualized in scatter mode using the 488 nm laser and standard instrument settings in fluorescence mode with standard fluorescence settings. Imaging data was analyzed with the ZetaView software 8.05.10 (Particle Metrix).

To determine the concentration and size distribution of EVs in the purified samples, NTA was performed using a ZetaView TWIN^® apparatus (Particle Metrix GmbH, Inning am Ammersee, Germany). Standard EV preparations from plasma of healthy individuals or conditioned media (CM) from the K562 cell line (HansaBiomed Life Sciences, Tallinn, Estonia) were used as controls. Between each sample passage, the counting chamber was thoroughly flushed with 0.1 µm filtered PBS. EV samples were diluted in 0.1 µm filtered PBS to an appropriate concentration before analysis and videos of the light-refracting particles were recorded with the following settings: 25 °C fixed temperature, 11 positions, 1 cycle, sensitivity 80, shutter 100, min. brightness: 30; min. area: 10; max. area: 1000, 3 measurements per sample. After capture, the videos were analyzed by the in-build ZetaView Software 8.05.10 (Particle Metrix GmbH, Inning am Ammersee, Germany).