Nano flow analysis

Exosomes were isolated using ExoEasy Maxi Kit (Qiagen, 76064, USA). Briefly, 2 ml plasma samples filtered using a 0.8 μm filter syringe (Millipore, SLAA033SB, USA) are mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with buffer XWP, eluted with 400μl Buffer XE (an aqueous buffer containing primarily inorganic salts). The exosome samples were stored at −20°C until further detection. Exosomes were observed by transmission electron microscopy (TEM) (FEI, Tecnai Spirit TEM T12). The size and concentration of the exosomes were assessed using Flow NanoAnalyzer (NanoFCM, Xiamen, China). Exosomal proteins CD81 (BD, 551108, USA) and CD9 (BD, 555317, USA) expression were measured using Nano-Flow analysis (NanoFCM, Xiamen, China).

Samples of 20 μl exosome were diluted to 90 μl. Approximately 30 μl diluted exosomes were added to 20 μl fluorescein-labeled antibodies (CD9, CD63, IgG). The samples were mixed and incubated at 37°C for 30 min in the dark. The samples were added with 1 ml PBS, and then centrifuged at 4°C, 110,000g for 70 min. Then the supernatant was discarded, the samples were added with 1 ml PBS, and then re-centrifuged at 11,000g for 70 min. The supernatant was removed and 50 μl PBS was used to suspend samples. Exosomal proteins CD9 and CD63 expression were measured using Nano-Flow analysis (NanoFCM, Xiamen, China).