2.3. Characterization of EV Fractions by DLS, NTA and TEM

The quality of all vesicle preparations was controlled according to the recommendations of the MISEV guidelines 2018, specifically, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM) were performed [4]. DLS was carried out using the Nano-flex 180 DLS system (Particle Metrix, Meerbusch, Germany), according to the manufacturer’s instructions. In brief, 20 μL of undiluted sample was placed on the measuring probe. Each sample was measured 5 times for 60 s and the results were averaged. Results were transferred from Intensity mode to Volume mode. The setup was as follows: Refractive Index 1.33; Min Size 0.8 nm; Max Size 6540 nm; Viscosity: Low Temperature 1.002; High Temperature 0.797; Transparency “on”; Shape: spherical. Estimation of the particles’ size using DLS was done with each of the fractions, including EV5, EV12, EV120 and fc. The particle concentration of these fractions was determined by NTA using the Zeta View system PMX110 (Particle Metrix, Meerbusch, Germany). According to the manufacturer’s protocol, samples were diluted with 10 mM HEPES 1:500 and one mL of diluted sample was injected into the NTA. The size distribution was measured in scatter mode and Zeta potential was measured in the Zeta Potential mode using the following setup: Sensitivity 85; Min size 20 nm; Max size 1000 nm; Min Brightness 10. Images were recorded at 11 positions and 5 cycles with a camera sensitivity of 65–81% and temperature monitored manually, ranging from 21 to 22 °C. As a negative control 10 mM HEPES solution was used. The particle number measured in the control was subtracted from the particle numbers measured in the samples. All measurements were performed in triplicate, and the mean value was calculated. The EV120 and fc fractions were additionally characterized by TEM. A 10 µL sample was loaded on a 300-mesh copper grid and fixed with 1% glutaraldehyde. Next, it was washed with double distilled water and negatively stained with 10 µL drop of 1% uranyl acetate. The images were taken using an electron microscope (LEO 906 E, Zeiss, Oberkochen, Germany) using SIS software (Olympus, Hamburg, Germany).