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Figure 2. hASC-EVs and hAMSC-EVs characterization. (A) EV size distribution by NanoSight particle tracking analysis for both hASC-EVs (blue dots) and hAMSC-EVs (orange dots). Plots show merged data of the three donors for each MSC type. (B) Transmission electron micrographs of EVs showing particles with characteristic cup-shaped morphology. Representative donors are shown. (C) The resolution of the FITC-fluorescent reference nanobeads (160, 200, 240, and 500 nm) indicates the flow cytometer performance in light scattering at default settings. After flow cytometer calibration, CFSE-stained EVs can be identified and gated in the FITC channel (FITC+ gate) vs. unstained particles. After gating, with respect to Ab-unstained samples, both hASC-EVs and hAMSC-EVs showed the presence of EV-defining molecules CD63 and CD81, while CD9 staining gave a very weak signal. Both hASC-EVs and hAMSC-EVs were also positive for MSC markers CD73 and CD90. CD44 labeling allowed a complete peak shift of the population, although without a sharp peak separation. Representative cytograms are presented. (D) Percentage of positivity for analyzed markers obtained averaging the three donors. CFSE: Carboxyfluorescein succinimidyl ester; EVs: extracellular vesicles; FITC: fluorescein isothiocyanate; hASCs: adipose-derived mesenchymal stromal cells; hAMSCs: amniotic membrane-derived mesenchymal stromal cells.