Streptavidin agarose ultralink resin

For total exosome purification from plasma, 2 mL of plasma were obtained from 5 mL of blood and diluted with 10 mL of PBS. Next, the same procedure used for exosome purification from cultured cells was followed. To enrich ADEs, total exosomes were resuspended in 0.35 mL of PBS and incubated for 60 min at room temperature with 1.5 μL of mouse anti-human glutamine aspartate transporter (GLAST) biotinylated antibody (Miltenyi Biotec, Auburn, USA) in 50 μL of 3% BSA per tube with mixing, followed by addition of 10 μL of streptavidin-agarose UltraLink resin (Thermo Fisher Scientific, Waltham, USA) in 40 μL of 3% BSA, and incubated for 30 min at room temperature with mixing. After centrifugation at 800×g for 10 min at 4 °C and removal of the supernatant, each pellet was resuspended in 100 μL of cold 0.05 M glycine-HCl (pH 3.0) by gentle mixing for 10 s and centrifuged at 4000×g for 10 min, all at 4 °C. Supernatants were then transferred to clean tubes containing 25 μL of 10% BSA and 10 μL of 1 M Tris-HCl (pH 8.0) and mixed before addition of 700 μL QIAzol Lysis Regent. Subsequently, ADEs RNA extraction was conducted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany).

Total EVs were isolated from frozen serum aliquots and enriched for neuronal-derived EVs as previously described^{22 (link)}. Briefly, EVs were isolated from 0.5 mL of frozen human serum following ExoQuick manufacturer instructions (System Biosciences, Cat # EXOQ5TM-1, Palo Alto, CA, USA) and MISEV2018 reporting guidelines⁴⁰ as previously described^{41 (link)}. For EV characterization data, see supplementary material and supplemental fig. S1. Neuronal-derived EVs were enriched by using 4 μg of biotinylated antibodies against neuronal surface markers CD171 (L1CAM) (clone 5G3; eBioscience, San Diego, CA, USA) in 50 μL of 3% Bovine Serum Albumin (BSA) (Thermo-Fisher Scientific Inc., Rockford, IL, USA), and followed by adding 15 μL of Streptavidin-agarose Ultralink resin (Thermo-Fisher Scientific Inc., Rockford, IL, USA) in 25 μL of 3% BSA per tube. The resin pellet was resuspended in 200 μL of 0.1 M glycine–HCl and centrifuged at 4 °C (for 10 min at 4500 × g). Supernatant fluid was then harvested to new collecting tubes containing 25 μL of 10% BSA and 15 μL of 1 M Tris–HCl and mixed. Neuronal-derived EVs were lysed with equal volume of mammalian protein extraction reagent (M-PER) (Thermo-Fisher Scientific Inc., Rockford, IL, USA), and then the lysis was ready for downstream analysis.