Isolation and Characterization of Extracellular Vesicles

ESCC cells were placed in Roswell Park Memorial Institute (RPMI) 1640 medium containing EVs-free 10% fetal bovine serum (FBS) and cultured for 3 d. Then, the cell supernatant was collected and centrifuged to remove cell debris. The EVs were extracted using Hieff™ Quick exosome isolation kit (41201ES50, YEASEN, Shanghai, China). The supernatant and exosome separation reagent were added into the Eppendorf (EP) tube with a proportion of 2:1 overnight, and then centrifuged at 100,00 g for 1–2 h and the precipitate was resuspended in phosphate buffered saline (PBS). The EV suspension (30 μl) together with an equal volume of radioimmunoprecipitation assay (RIPA) lysis buffer was continuously dissolved in microwave (2 times, 10 s/time). Then, the EVs were purified at 4°C at 12,000 × g for 2 min, and the supernatant was retained and stored at −80°C. Protein quantification of EV was performed by bicinchoninic acid (BCA) kit (Beyotime, Nantong, China).
EVs identification: Transmission electron microscope (TEM; JEM-1010, JEOL, Tokyo, Japan) was adopted to observe EV morphology, tunable resistive pulse sensing (TRPS) to examine concentration and diameter of EVs, and Western blot analysis to test EV markers (CD81 and CD9) and non-marker protein (Calnexin and GM130) (Li Z et al., 2019 (link)).