Zetaview analyzer

Extracellular vesicle preparations from HSC-3 and OSC-20 cell line supernatants were analyzed for size by nanoparticle tracking analysis with a Zetaview analyzer (Particle Metrix GmbH, Meerbusch, Germany) following the manufacturer’s recommendations.

NVs (10 μg/mL) were dispersed in PBS, and then the particle concentration of NVs was assessed by ZetaView analyzer (Particle Metrix GmbH, Meerbuch, Germany). Measurements were evaluated in triplicates, and each individual data was acquired from two stationary layers with five times measurements in each layer. Sensitivity of the camera was configured at 70 in all measurements. Data were analyzed using ZetaView analysis software version 8.2.30.1.

When TSCs reached 80% fusion, the medium was replaced with a medium containing 10% serum without exosomes. After 24 h, the culture medium was collected and centrifuged sequentially at 300×g for 10 min, 3000×g for 10 min, and then 10,000×g for 30 min to remove cellular debris. The supernatants were then ultracentrifuged at 100,000×g for 2 h to precipitate the exosomes, which were then resuspended in 200 μL of PBS. The total protein concentration of exosomes was quantified using the bicinchoninic acid assay (Beyotime, Shanghai, China). Morphology and quality of exosomes were examined by transmission electron microscopy (TEM), and particle size was measured using a ZetaView Analyzer (Particle Metrix, Germany). Western blot was used to detect exosome surface markers CD9, TSG101, and Hsp70. The above experiments were repeated three times.

EV and NV were dispersed in PBS at the optimal concentration for measuring, and then particle concentrations were measured using ZetaView analyzer (Particle Metrix GmbH, software version 8.2.30.1). Measurements were assessed in duplicate, and each individual data set was obtained from two stationary layers with 11 measurements in each layer. Camera sensitivity was set at 80% in all measurements. For transmission electron microscopy (TEM), formvar/carbon-coated copper grids (Ted Pella, Inc., Redding, CA, USA) were glow discharged before the samples were loaded. The grids and samples were incubated for 15 min before being fixed in 2% paraformaldehyde and in 2.5% glutaraldehyde with PBS washes in between. The samples were then washed in dH2O and then contrasted in 2% uranyl acetate. The preparations were examined using a LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany).

OMVs (5 μg/mL) were dispersed in PBS, and then the OMVs particle concentration were assessed by ZetaView analyzer (Particle Metrix GmbH). Measurements were assessed in triplicates and each individual data was obtained from two stationary layers with five times measurements in each layer. Sensitivity of camera was configured at 70 in all measurements. Data were analyzed using ZetaView analysis software version 8.2.30.1.

SyEV were diluted in PBS, and the number of vesicles was determined using ZetaView analyzer (Particle Metrix GmbH, Meerbuch, Germany). The analyses were performed in triplicate, and each data point was obtained from two stationary layers with five measurements in each layer. The sensitivity of the camera was adjusted to 70 in all measurements. Data were interpreted using ZetaView analysis software version 8.2.30.1.