Isolation of exosome, labeling, and quantification

A total of 2 mL of serum or 10 mL of supernatant was mixed with exoEasy maxi kit exosome precipitation solution to isolate exosomes, and exosome isolation was performed as per the manufacturer’s instructions (QIAGEN, Hilden, Germany). 2.5 μg/mL DiI (Yeasen Biotechnology, Shanghai, China) was added to label exosomes before the mixture was centrifuged for 1 h. The result was imaged by a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). The FluoroCet ultrasensitive exosome quantitation assay kit (System Biosciences, Palo Alto, CA, USA) was used for the quantification of exosomes to guarantee that in each experiment the same amount of exosomes was used. As mentioned above, 5 × 10⁸ exosomes were used for extraction of exosomal RNA, and 1 × 10⁸ exosomes were used for in vitro experimentation with exosomes stimulation.