Zetaview

hADSCs at passage 3 were cultured under hypoxia (5% oxygen) and normoxia (20% oxygen) until cells reached 70–80% confluency. The medium was then replaced with serum-free Dulbecco’s modified Eagle’s medium (DMEM)/F12 for 24 h to collect conditioned medium. Cell debris and apoptotic bodies were discarded after sequential centrifugations at 500 ×g, 3,000 ×g and 10,000 ×g for 5, 15 and 60 min, respectively. The supernatant was then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h using a 45 Ti rotor (Beckman Coulter, Optimal L-80XP, USA). The remaining precipitate was resuspended in PBS and filtered with a 0.22-mm filter (Millipore, Billerica, MA, USA), then ultra-centrifuged at 100,000 ×g at 4 °C for 1 h. EVs were stored at −80 °C until further use. EVs obtained from both conditions were characterised using 80 kV TEM (HITACHI H-7000FA, Japan). The particle size distribution and concentrations of EVs were analysed by ZetaVIEW (Particle Metrix, ZetaVIEW S/N 17–315, Germany) and Nanoparticle Tracking Analysis software (ZetaVIEW 8.04.02). Antibodies against CD9, CD63, Alix, β-actin and calnexin (Abcam, London, UK) were used in western blotting. In addition, the supernatant of EV lysates was prepared, and the protein content was evaluated using a BCA Protein Assay Kit (Sigma, Silicon Valley, USA).

Isolated exosomes were diluted in PBS and analyzed using the ZetaView (S/N 17-310, Particle Metrix, Germany) with NTA software (ZetaView 8.04.02). Triplicate measurements were recorded for each sample. Size distribution and concentration profiles were averaged to derive the representative size distribution profiles.

After the isolation, the concentration of all the samples was measured b) by ZetaView (Particle Metrix GmbH, Germany). 1 μL of concentrated EVs was diluted in sterile-filtered PBS in a dilution 1:1,000 and visualized using the ZetaView (sensitivity 80%, shutter 100, 11 positions, 2 cycles; Particle Metrix, Germany).

EVs were quantified in media collected from calcification assays using previously established protocols [53 (link)]. Briefly, media was aspirated from VSMCs and spun at 500× g for 5 min to eliminate cell debris and snap-frozen until further analysis. Further, calibration beads and 50 uL of the supernatants were diluted in PBS to a final volume of 1 mL. EVs quantification was performed using nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix, Inning am Ammersee, Germany). Optimum scanning conditions were established using previously described protocols [53 (link)]. A washing step with distilled water was performed between each technical sample reading. Data analysis was performed with NTA 2.1 software (ZetaView, Particle Metrix, Inning am Ammersee, Germany).

Nanoparticle quantification was carried out as previously described (Juodeikis et al., 2022 ). Briefly, particles were quantified using the ZetaView instrument (Particle Metrix) with ZetaView (version 8.05.12 SP1) software running a 2 cycle 11 position high frame rate analysis at 25°C. Camera control settings: 80 Sensitivity; 30 Frame Rate; 100 Shutter. Post‐acquisition parameters: 20 Min Brightness; 2000 Max Area; 5 Min Area; 30 Tracelength; 5 nm/Class; 64 Classes/Decade.