Gatan microscopy suite version 3

EVs were collected from GDNF-transfected macrophage media, and the size and number of particles were evaluated using NanoSight 500, Version 2.2 (Wiltshire, UK). In addition, the concentration, and zeta potential (ZP) of the EVs were measured using a ZetaView QUATT Nanoparticle Tracking Microscope PMX-420 (Particle Metrix, Inning am Ammersee, Germany). For this purpose, the EVs were diluted to a concentration of about 2 × 10⁷ particles/mL, with 20 nm of filtered PBS. Measurements were performed at 11 positions using the following settings: maximum area 1000, minimum area 5, minimum brightness 20, camera level 16, and threshold level 5. Total protein was calculated using standard BCA assay. The AFM imaging was performed as described earlier [42 (link)]. A drop of isolated EVs in 50 mM phosphate buffer, pH 7.4, at total protein 10 µg/mL was placed on a glass slide and dried under an argon flow. For TEM imaging, EVs were adsorbed onto Formvar coated copper grid (400 mesh), stained with 2% uranyl acetate for 1 min. The samples were observed using a JEOL JEM-1230 transmission electron microscope operating at 80 kV (JEOL USA INC., Peabody, MA, USA) and images were taken using a Gatan Orius SC1000 CCD camera with Gatan Microscopy Suite version 3.10.1002.0 software (Gatan, Inc., Pleasanton, CA, USA).

EVs were visualized by negative-stain transmission electron microscopy (TEM) in the UNC Microscopy Services Laboratory. Briefly, this involved floating a glow-discharged formvar/carbon-coated 400 mesh copper grid (Ted Pella, Inc., Redding, CA, USA) on a 20μl droplet of the sample suspension for 12 min. This was transferred quickly to 2 drops of deionized water followed by addition of a droplet of 2% aqueous uranyl acetate stain for 1 minute. The grid was blotted with filter paper and airdried. Samples were then visualized at 80kV using a JEOL JEM-1230 transmission electron microscope operating (JEOL USA INC., Peabody, MA, USA). Images were taken using a Gatan Orius SC1000 CCD camera with Gatan Microscopy Suite version 3.10.1002.0 software (Gatan, Inc., Pleasanton, CA, USA).

To visualize EVs, isolated EVs were prepared for negative-stain transmission electron microscopy. A glow-discharged formvar/carbon-coated 400 mesh copper grids (Ted Pella, Inc., Redding, CA) was floated on a droplet of the sample suspension for 12 minutes, transferred quickly to 2 drops of deionized water followed by a droplet of 2% aqueous uranyl acetate stain for 1 minute. The grid was blotted with filter paper and air-dried. Samples were observed using a JEOL JEM-1230 transmission electron microscope operating at 80kV (JEOL USA INC., Peabody, MA) and images were taken using a Gatan Orius SC1000 CCD camera with Gatan Microscopy Suite version 3.10.1002.0 software (Gatan, Inc., Pleasanton, CA).