Exosome isolation and characterization

Exosomes were isolated from the culture medium of FHC, HCT116 and DLD1 cells by using an ExoQuick TC kit (SBI, USA), and exosomes from the plasma of subjects were purified by using an ExoQuick Plasma Prep with Thrombin kit (SBI, USA). The size distribution of the exosomes was characterized by nanoflow cytometry using a U30 Flow NanoAnalyzer (NanoFCM, Inc., China) with technical assistance provided by KeyGEN Biotech Co. Ltd. (Jiangsu Province, China). The shape and size of the exosomes were observed by transmission electron microscopy (TEM) (Tecnai G2, FEI, USA). Moreover, the characterization of the exosomes was confirmed by the presence of exosomal protein markers TSG101 (# ab125011, Abcam, USA) and Alix (# 92880, CST, USA). The green fluorescent dye PKH67 (Umibio, China) was utilized to label exosomes isolated from the culture medium of cells. After dye was incubated with recipient cells for 3 h, fluorescence microscopy (Zeiss, Germany) was performed to visualize PKH67-labelled exosomes in recipient cells. The detailed procedures were described in our previous study [27 (link)].