Carbon coated 400 hex mesh copper grid

Following EV isolation, exosome-enriched isolates were resuspended in 50–100 µL of sterile filtered PBS for electron microscopy imaging according to Lasser et al. [61 (link)]. Briefly, intact exosomes (5 µL) were dropped onto Parafilm for each sample preparation. With forceps, a carbon-coated 400 Hex Mesh Copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) was positioned with coating side down on top of each drop for 60 min. Grids were then washed with sterile filtered PBS three times, and the grid was transferred on top of a 30 µL drop of PBS each time. Excess solution was wicked off the grid gently using absorbing paper between each wash, while avoiding direct contact with the coating side. Exosomes were fixed by positioning the grid on top of a 20-µL drop of 2% PFA (EM grade, Electron Microscopy Sciences) for 10 min. After washing, grids were then transferred on top of a 20 µL drop of 2.5% glutaraldehyde (EM grade, Electron Microscopy Sciences) for 10 min, followed by three washes with particle-free filtered water. Grid samples were stained on a 20 µL drop of 2% uranyl acetate (EMS grade) for 10 min before embedding on 20 µL of 0.13% methyl cellulose and 0.4% uranyl acetate for another 10 min. Excess liquid was removed using absorbing paper and grids were left to dry coated side up before imaging on the electron microscope.

TEM was performed to confirm the morphology of EVs according to Lasser et al. [62 (link)] and our previous publication [20 (link)]. Briefly, EV isolates were resuspended in 50–100 μL of sterile filtered PBS. For each sample preparation, intact EVs (5 µL) were dropped onto Parafilm. A carbon-coated 400 hex mesh copper grid (Electron Microscopy Sciences, EMS) was positioned using forceps with the coating side down on top of each drop for one hour. Grids were washed with sterile filtered PBS three times and then the EV samples were fixed for 10 min in 2% PFA (EMS, EM Grade). After washing, the grids were transferred on top of a 20 µL drop of 2.5% glutaraldehyde (EMS, EM Grade) and incubated for 10 min at room temperature. Grid samples were stained for 10 min with 2% uranyl acetate (EMS grade). Then, the samples were embedded for 10 min with 0.13% methyl cellulose and 0.4% uranyl acetate. The coated side of the grids were left to dry before imaging on the CM120 Biotwin electron microscope [62 (link)].