Nanoparticle Tracking Analysis

The biophysical properties (number and size) of EVs were determined using Nanoparticle Tracking Analysis (NTA) with a NanoSight NS300 instrument (Malvern Analytical), employing the following settings: camera level 7 and screen gain 10. Briefly, EVs were isolated from 100 μL of precleared plasma through ultracentrifugation and then resuspended in 1 mL of sterile dPBS. A 1:100 dilution was prepared by adding 10 μL of the resuspended pellet to 990 μL of sterile dPBS. Using a 1 mL syringe (Henke Sass Wolf #4010-200V0), the diluted sample was slowly pushed through the fluid lines, and videos of the samples were recorded for 60 seconds each for subsequent image analysis. To ensure accuracy, three technical replicates were generated for each sample by advancing 100 μL of the sample between each recording.