2.2. Transmission Electron Microscope (TEM)

TEM analysis was used to confirm the presence of DPC-EVs in the conditioned medium. For fixation, equal volumes of EVs and 4% paraformaldehyde were absorbed by formvar carbon-coated EM grids for 30 min and washed twice with PBS for 3 min. The grid was rinsed 4 times with 50 mM glycine in PBS for 3 min, then blocked with Blok-Ace for 30 min and incubated with anti-CD63 for 1 h at 1:100 ratio. The antibody was washed with PBS and 0.05% Tween-20 (T-PBS) for 3 min and treated with 10 nm gold particles conjugated with a secondary antibody for 30 min, followed by incubation with 1% glutaraldehyde for 5 min. After washing with T-PBS several times, the EVs were observed by TEM at 80 kV. For the negative control, all the above procedures were followed except for primary antibody incubation. EVs were only incubated with 1% glutaraldehyde for 5 min. EVs size distribution or diameter was measured by ImageJ software (public domain free software version: java 1.8.0_172) after capture of EV images by TEM. Total Area(A) of EVs was measured upon nanometer (nm) scale. Later, the following equation was used for EVs diameter (D = nm) analysis.