300 mesh copper grid support film

The isolated products from each experiment were processed in the same way. Paraformaldehyde was first added to the sample with a final concentration of 4% (w/v) and then incubated for 20 min at room temperature. A 100-μl droplet of isolated exosome sample was then placed on a sheet of Parafilm (VWR, USA). A 300-mesh copper grid support film (Electron Microscopy Sciences, USA) was placed on the droplet with membrane side down to allow the sample absorption for 20 min. Then, this grid was transferred to a 100-μl droplet of distilled water for 2 min. This process was repeated three times. This grid was then transferred to a 100-μl droplet of uranyl-acetate solution for negative staining for 8 min. Last, the grid was rewashed using distilled water and left to dry at room temperature. The sample was finally observed under TEM (FEI Tecnai G2 Twin, FEI Company, USA).

TEM validation follows our previous procedure.^{65 (link)} 4% paraformaldehyde (Sigma-Aldrich, St. Louis,
MO) was used for fixing the isolated samples. 100 μL droplets
of the fixed sample were covered with a 300-mesh copper grid support
film (Electron Microscopy Sciences, Hatfield, PA) for absorption.
The grids were washed with distilled water and then stained using
uranyl acetate solution (Electron Microscopy Sciences). The grids
were washed with distilled water again and dried at room temperature.
An electron microscope (FEI, Hillsboro, OR) was used for observation.

Particle size of EVs was measured by Nanosight LM10 system (Malvern, UK) and qNano system (Izon Science, UK). All samples were diluted in PBS to a final volume of 1 mL. Ideal measurement concentrations were found by pretesting the ideal particle per frame value (20-100 particles/frame) on Nanosight instrument. The concentration of each sample was incrementally increased until the count rate reached a medium value at the highest laser attenuator value for tRPS measurements. For transmission electron microscope (TEM) imaging, the isolated EVs were mixed with paraformaldehyde at a final concentration of 4% wt/vol. After incubation at room temperature for 20 min, a drop of sample (100 µL) was added on a sheet of parafilm. A 300-mesh copper grid support film (Electron microscopy sciences, PA, USA) was placed on the sample liquid drop (membrane side down) to allow the membranes adsorption for 20 min. Next, the grid was washed three-times in a 100 µL drop of distilled water for 2 min, and then was transferred to a 100 µL drop of uranyl acetate solution for negative staining for 10 min. After washing away with unstained uranyl acetate, the grid was left to air-dry at room temperature. And the sample was observed under JEM1400 electron microscope (JEOL, Tokyo, Japan).