Figure 1.

Characterization of Exoquick-precipitated plasma exosomes. (A–C) Atomic Force Microscopy (AFM). (A) Representative AFM micrograph (2.5 μm × 2.5 μm) of mBC exosomes enriched by ExoQuick, diluted in PBS and deposited on a mica substrate. (B) Height profile of a single vesicle (blue line in panel A) matching the typical lateral dimensions of exosomes (<150 nm). (C) Quantification of the exosome diameter: histogram show the distribution of the diameter of exosomes, as extracted by grain analysis (Gwyddion) of three distinct AFM images of the same sample. (D) Nanoparticle Tracking Analysis (NTA). Representative histogram showing the particle size distribution of an exosome preparation analyzed by Nanosight and diluted 1:1000 in water. (E–G) Western Blot. (E) After ExoQuick precipitation, samples were subjected to floatation on a linear sucrose gradient. Protein distribution in the gradient is shown as µg of proteins recovered in each fraction (grey line; scale on the right). Enlarged data (black line; scale on the left) are also shown. (F) Ponceau staining of the gel. (G) Cropped immunoblot showing CD9 immunoreactivity of the different recovered fractions (full length blot is displayed in Supplementary Fig. 1). (H) Scanning Electron Microscopy (SEM). Representative pictures, acquired at different magnification, of a sample whose CD9 positive fractions (fractions 3–5) were pooled and subjected to SEM analysis. (I) Dynamic light scattering (DLS) analysis. CD9 positive fractions 3–5 were pooled and analyzed by DLS. Histograms represent the size distribution of the vesicles. The profile shows the presence of distinct populations. The first one with an average size of about 30 nm and the second one of 110 nm. A population with an average diameter of 450 nm was also observable, probably due to protein aggregation induced by ExoQuick treatment.