Transmission electron microscope

In brief, a total of 5 × 10⁶ hAECs were cultured in a 100 mm culture dish. After 24 h, the complete culture medium was replaced with 10 mL of serum-free DMEM/F12 medium. After another 24 h, the CM from hAECs was collected and centrifuged at 3,000 g for 15 min to remove the dead cells and cellular debris. The supernatant was filtered through a 0.22 μm filter (Millipore, Billerica, MA, USA). Then, 10 mL supernatant was further concentrated by centrifugation for 30 min at 10,000 g in a pre-rinsed centrifugal filter tube (3 kDa; Amicon Ultra-15; Millipore) to approximately 1 mL. A 1:2 volume of total exosome isolation reagent (Invitrogen, Carlsbad, CA, USA) was added to the concentrated liquid and mixed well by vortexing. After an overnight incubation, the mixture was centrifuged at 10,000 g for 60 min to obtain the pellet. The pellet of exosomes was resuspended in PBS. All procedures were performed at 4°C. The exosomes were stored at −80°C or used for the experiments.
The ultrastructure of the exosomes was analyzed under a transmission electron microscope (Zeiss, Oberkochen, Germany). The protein levels of Alix, CD63, and CD9 (representative markers of exosomes) were detected using western blot. To determine the sizes of the purified vesicles, a nanoparticle tracking analysis (NTA) was performed using Zetaview software (Particle Metrix, Meerbusch, Germany).