Nanosight instrument

Exosome samples were imaged under a JEM-1400 Plus transmission electron microscope (JEOL Ltd., Tokyo, Japan) at an under focus of 0.8–1.5 μm and recorded using an UltraScan OneView CMOS camera (Gatan, Pleasanton, CA, USA). Samples were prepared by loading 5 μL solution onto an EM grid covered with glow-discharged continuous carbon film. The grid was washed with deionized water after 1 min and stained with 1% uranyl acetate for 1 min. After removal of staining solution using filter paper, the grid was dried completely in open air.
The size distribution of particles was determined by nanoparticle tracking analysis (NTA), which assesses the combined properties of light scattering and Brownian motion. Isolated EVs in liquid were diluted in 1 mL phosphate-buffered saline (PBS; Lonza), and visualized and counted by a Nanosight instrument (Malvern Instrument, Worcestershire, UK) at a temperature of 25 °C using a 488 nm laser.

To conduct TEM for exosomes morphology, the fixed exosomes in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) were moved onto the carbon/Formvar-coated grid (Ted Pella, Inc., Tustin, CA, USA) at room temperature for 30 min. After washing in PBS, the grid was cultured with 1% glutaraldehyde (Sigma-Aldrich), followed by the observation under JEOL JEM-2000EXII microscope (Jeol Ltd, Tokyo, Japan). To perform NTA for size distribution, exosomes were diluted to 10⁶ to 10⁹ particles/ml, then analyzed by NanoSight instrument (Malvern Panalytical, Malvern, UK).

Exosomes were harvested from mice serum or conditional medium of cultured C2C12 myotubes. Mice sera were obtained from heart puncture. 0.5 ml serum from each mouse was diluted 5-times with PBS before isolation of exosomes. For purification and characterization of exosomes from serum or conditional medium, cell debris and organelles were eliminated by centrifugation at 1,000 g for 10 min, 4°C. The supernatant fraction was further centrifuged at 16,000 g for 30 min. The second supernatant was sterile filtered through a 0.22 μm filter. Exosomes were pelleted at 120,000 g for 90 min at 4°C (L8-70M ultracentrifuge, Beckman-Coulter, Indianapolis, IN, United States). Finally, the exosome pellet was re-suspended in 100-400 μl RNA stabilization reagent (Qiagen, Germantown, MD, United States) for RNA extraction or sterile PBS for other experiments. Exosomal size and concentration were verified using a NanoSight instrument (Malvern, Westborough, MA, United States) and an exosome marker (TSG101) was assessed by Western blot (Su et al., 2018 (link)) and exosome images taken by electro-microscopy (Supplementary Figure 3). Exosomes were also isolated from serum of vehicle (0.01% DMSO) injected mice. There are no significant differences in exosome size and concentration between vehicle injected and non-injected mice (Supplementary Figure 4).