NTA is widely used in vesicle research for the characterization of the size and concentration of EVs (27 ). With NTA, the Brownian motion of individual particles in solution is tracked based on their light scattering. Variables include the camera level, detection threshold, viscosity, temperature, and the dilution of the sample. There are some attempts to standardize this method with regard to characterization of EVs (27 , 32 (link)). An NS500 (NanoSight Limited, London, UK) equipped with an electron multiplying charge coupled device camera (Andor Technology, Tokyo, Japan) and a 405 nm laser was used. During measurements, temperature was kept at 22°C. The viscosity of water at 22°C (0.95 mPa s) was used, as samples were diluted several fold in PBS buffer. Silica beads with a diameter of 100 nm and known concentration were used to adjust the focus height of the objective and to calibrate the concentration (27 ). NTA v2.3.0.17 software (NanoSight Limited) was used for data analysis. Before the measurement, samples were 500,000-fold diluted with PBS buffer. Ten videos of 30 seconds were captured per measurement at camera level 15. The detection threshold was set at pixel value 16.
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