NTA was performed on NanoSight LM14 system (NanoSight Technology, Malvern, Worcestershire, UK) equipped with a 532 nm green laser for determining the EV concentration and size distribution. Briefly, 10 μL separated EV samples were diluted with freshly 0.22 μm filtered PBS and loaded into the detection chamber by syringe. The same settings were used: camera level: 11, detect threshold: 14, capture: 60 s, number of capture: 5, temperature: 25 °C. The data were processed by NTA software (NTA version 3.3; Malvern Instruments, Malvern, Worcestershire, UK). A series of known numbers of 100 nm silica microspheres (Polysciences, Inc, catalog No: 24,041–10, Warrington, PA, USA) were used for the calibration of our measurement before using any EV samples. EV isolates from three kits were diluted to the optimum NTA detection range (1–10 × 108 particles/mL) before measurement.32 (link),33 (link) The same Nanosight parameters were used for all samples for a consistent comparison. The original particle concentrations from the isolates were then calculated based on the measured concentrations and the dilution factor.