Nanovan

TEM was performed on 3T3-L1–derived EVs (Ad-EVs) and SAT-EVs isolated by ultracentrifugation, resuspended in PBS 1×, placed on 200 mesh nickel formvar carbon-coated grids (Electron Microscopy Science), and left to adhere for 20 minutes. Grids were then incubated with 2.5% glutaraldehyde, containing 2% sucrose, and EVs were negatively stained with NanoVan (Nanoprobes), after being washed in distilled water, and observed under a Jeol JEM 1010 electron microscope.

For each sample, 1 ml of plasma was thawed on ice and centrifuged at 2,000 × g at 4°C for 40 min to remove platelet contamination. EVs were then precipitated as previously described (21 (link)). After precipitation, EVs were resuspended in 150 μl of Roswell Park Memorial Institute (RPMI) medium supplemented with penicillin, streptomycin, and amphotericin B, plus 10% of dimethyl sulfoxide (DMSO), and stored at -80°C until use. EV size and concentration were assessed by nanoparticle tracking (NTA) analysis (21 (link)). The presence of EVs on precipitated samples was confirmed by transmission electron microscopy. EVs were left to adhere to 200-mesh Nickel Formvar^® carbon-coated grids (Electron Microscopy Sciences) for 10 min. Grids were then washed with phosphate-buffered saline (PBS), fixed with 2.5% glutaraldehyde containing 2% sucrose, negatively stained with NanoVan^® (Nanoprobes), and observed by JEOL JEM-1400 Flash electron microscope (Tokyo, Japan). The presence and percentage of exosomes in our precipitated EV samples were measured by flow cytometry using CD9 and CD81 phycoerythrin (PE)-conjugated antibodies (Figure 1).