Extracellular Vesicle Isolation and Characterization

EVs from P17 and P20 UFs were isolated with the addition of exosome precipitation solution (Exo-Quick-TC, System Biosciences, Mountain View, CA, USA), which were incubated overnight at 4 °C according to the manufacturer’s instructions. The UFs with Exo-Quick-TC were then centrifuged at 1500 x g for 30 min at 4 °C. EVs were suspended and their protein concentration adjusted either in PBS (1 µg/µl) for TEM analysis and in vitro culture experimentations, whereas EVs were suspended in mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific, Waltham, MA, USA) for western blot analysis^{26 (link),27 (link)}.