fig1

Figure 1. Characterization of brain endothelium-derived extracellular vesicles. Vesicles were isolated from endothelial cell cultures treated with Aβ₄₀ or PBS. TEM demonstrates average size and spherical morphology of (A) control EVs and (B) EV-Aβ₄₀. NTA of isolated EVs loaded with (A) PBS as a vehicle control and (B) Aβ₄₀ shows variable diameter distribution in the nanometer range. Red outlines indicate error bars (± SEM) from five 15-s videos of NTA. (C) ELISA for Aβ₄₀ indicates effective enrichment of EVs isolated from medium of endothelial cells treated with Aβ₄₀. Student’s unpaired t-test, ****P < 0.0001 (isolated vesicle lysates from individual cell culture dishes; n = 3). (D) Western blot of lysed EVs for tetraspanin markers CD9, CD63, CD81, and Aβ₄₀. CD31 expression confirmed that the EVs were secreted from an endothelial cell line. (E) Fluorescent images of EVs isolated from endothelial cells transfected with CD9-RFP (red) and exposed to fluorescent Aβ₄₀ (green). DAPI indicates genetic material (blue). Fluorescent intensity plot shows co-localization of CD9 vesicle marker (red) and Aβ₄₀ (green) in the EV-Aβ₄₀ group. Scale bar for representative images is 10 µm and zoom inserts are 30 µm. White arrow indicates the region of interest for the zoom inserts. TEM: Transmission electron microscopy; EV: extracellular vesicles; NTA: nanoparticle tracking analysis; SEM: standard error of the mean; ELISA: enzyme-linked immunosorbent assay.