Quantifying Exosome Particle Size and Concentration

Particle size and concentration of isolated exosomes and negative controls were quantified using Nanoparticle Tracking Analysis (Malvern Instruments Ltd.; NanoSight NS300) as per manufacturer’s instructions at the Structural and Biophysical Core Facility, The Hospital for Sick Children (Toronto, Ontario, Canada). NTA uses the properties of Brownian motion and light scattering to measure particle size and concentration (particles/mL) of EVs. The software tracks individual particles frame by frame and calculates particle size based on Stokes-Einstein equation [64 (link)]. A 1:700 dilution factor for bovine samples and a 1:300 dilution factor for human samples were used for the analysis. Standard curves ranging from 1:100 to 1:700 (v/v in 1X-filtered PBS) were run per species to determine the optimal dilution range for the exosomes (60–100 particles/frame). An absolute control of 1X-filtered PBS was also assessed to test for the purity of the reconstitute media. Settings: detection threshold of 8, camera level of 15, 3 replicates of 30 s captures, and a blue laser 488 nm.