Em 2010 transmission electron microscope

Exosomes were prepared using differential ultra-centrifugation as follows: Plasma (5 ml) was centrifuged at 1,600 × g for 20 min at 4°C and at 10,000 × g for 30 min at 4°C. The supernatant was filtered through 0.22-µm disposable filter units (Merck KGaA, Darmstadt, Germany) and centrifuged at 100,000 × g for 2 h at 4°C. Subsequent to washing with 10 ml PBS (Invitrogen; Thermo Fisher Scientific, Inc.), the exosomes were suspended in 0.1 ml PBS and stored at −80°C until required. The diameter of the exosomes was determined using a Zetasizer Nano (Malvern Instruments China, Shanghai, China) according to the manufacturer's protocol. The purified exosomes were observed using an ×250,000 magnification EM-2010 transmission electron microscope (JEOL, Ltd., Tokyo, Japan).