Fei tecnai t20 transmission electron microscope

After the cells had reached a confluency of 70–80%, the medium was changed to a RPMI 1640 medium with 10% exosome-depleted FBS (obtained through ultracentrifugation at 120,000×g at 4 °C for 6 h [18 (link)]). After 48 h, 50 ml of the conditioned medium (CM) was collected from each cell line, and ultracentrifugation was used to extract exosomes from the medium, following previously described standard procedures [19 ]. In order to collect blood samples for serum exosome isolation, ethylenediaminetetraacetic acid (EDTA) containing collection tubes were used. Within an hour, the tubes were centrifuged at 1900×g at 4 °C for 10 min, using a swinging bucket rotor. A new tube was used to collect the upper (yellow) serum phase, and 16,000×g centrifugation at 4 °C for 10 min was conducted to eliminate additional cellular fragments, as well as cell debris. Then, an exoEasy Maxi Kit (Qiagen, Hilden, Germany; Cat. Number: 76064) was used, as instructed by the manufacturer, to isolate serum exosomes. As described in a previous study [20 (link)], a FEI Tecnai T20 transmission electron microscope (TEM) (FEI Company, USA) was used to observe the exosomes, while a Nano Sight NS 300 system (Nano Sight Technology, Malvern, UK) was used to determine exosome quantity and size.