Figure 3: Identification and characterization of FRα-positive exosomes in specimen of human CSF.

(a) Sequential centrifugation steps were conducted with human CSF specimen and the resulting pellets were analysed by western blotting. P10 refers to the pellet from 10,000 g centrifugation step; P100 refers to the pellet from 100,000g centrifugation step. The input and the remaining supernatants after the 100,000 g centrifugation were concentrated 20-fold prior loading on the gel. FRα and the exosomal marker Flot-2 are enriched in P100, whereas transferrin remains mainly in the supernatant. Full-scan images are displayed in Supplementary Fig. S6. (b) A 100,000 g pellet was loaded on top of a discontinuous sucrose density gradient and ultracentrifuged for 16 h. The fractions were then analysed by western blotting. FRα is enriched in the same fractions as Flot-2. Full-scan images are displayed in Supplementary Fig. S6. (c) For electron microscopy, a 100,000 g pellet was negatively stained with uranyl acetate and immunolabelled with antibodies against FRα or Flot-2. The pellet contains mainly small membrane vesicles with a diameter of about 40–100 nm, with the typical cup-shaped morphology of exosomes, that are positive for FRα and Flot-2. Scale bars, 200 nm. (d) High-resolution imaging of the 100,000 g pellet and colocalization analysis of FRα and the exosomal marker Alix by two-colour STED microscopy. Although standard confocal microscopy fails to display the details, STED microscopy reveals the typical size of exosomes and analysis of the images demonstrates a colocalization of FRα with Alix of 36%±8% (data calculated with the NIH Image J software). Scale bar, 1 μm. (e) STED microscopy allows exact size determination of the vesicles positive for FRα and Alix (arrowhead in d).