*2.3. NanoFCM Analysis*

For the Flow Nano Analyzer (NanoFCM Co. Ltd., Nottingham, UK) analysis, the system was calibrated using 200 nm polystyrene beads (NanoFCM Co. Ltd., Nottingham, UK) with a defined concentration of 5.7 × <sup>10</sup><sup>8</sup> particles/mL, which were also used as a

reference for the particle concentration. In addition, monodisperse silica beads (NanoFCM Co. Ltd., Nottingham, UK) of four different sizes were used as reference standards to calibrate the size of the EVs. Freshly filtered (0.22 μm) PBS was analyzed as a background signal, which was subtracted from the other measurements. EV samples were diluted with filtered PBS resulting in a particle count in an optimum range of 2500–12,000 events, and sample data were collected for 1 min with a sample pressure of 0.4 kPa. Particle concentration and size distribution were calculated using the NanoFCM software NF Profession v1.08) (NanoFCM Co. Ltd., Nottingham, UK). Median and interquartile range (IQR) were calculated using R software v4.1.0 (R Foundation, Vienna, Austria).
