Exocet exosome quantification kit

Plasma or culture supernatants were centrifuged at 1000 × g for 20 minutes to remove remaining cell debris. Total exosomes were isolated with Total Exosome Isolation (Invitrogen) as described in the manufacturer’s protocols. In brief, 200 μl plasma was diluted with 100 μl 1× PBS, and 60 μl of exosome precipitation reagent was added. For media exosome isolation, exosome precipitation reagent was mixed with 1 ml cell supernatants. The mixtures were vortexed briefly and incubated at room temperature for 10 minutes. Following the incubation, samples were centrifuged at 10000 × g for 10 minutes to pellet the exosomes. Exosomes were resuspended in 200 μl 1× PBS in a 37 °C incubator for ~1 hour. Purified exosomes were quantified using EXOCET Exosome Quantification Kit (System Biosciences).

dSH-SY5Y or hiPSCs cells were plated on coverslips in the respective serum-free growth medium. Before the uptake assay, exosomes were isolated from brain tissue or conditioned media of raSH-SY5Y cells and labelled with PKH67 or PKH26 as described above. In order to use equal amounts of exosomes in the cell cultures, exosomal protein content was QuantITated by using BCA (Bio-Rad) or QuantIT (Invitrogen). Brain exosome abundance was quantified according to the AChE activity (EXOCET Exosome Quantification kit; System Biosciences) according to the manufacturer’s protocol. The uptake was performed by incubating cell cultures with 100 µl of exosome solutions (corresponding to an exosomal protein content of 0.62 ± 0.28 μg/μl from brain or 0.71 ± 0.33 μg/μl from conditioned media; equal to 1.4e10 exosome abundance from brain) in a humid chamber for 3 h (37 °C, 5% CO₂). For inhibition experiments, cultured cells were pre-incubated for 30 min with the endocytosis inhibitors, dynasore (dynamin inhibitor, 80 µM), phenylarsine oxide (clathrin inhibitor 20 µM), genistein (caveolae inhibitor, 200 µM) all from Sigma-Aldrich. Isolated exosomes in PBS were added to cells for 3 h as above and flow cytometry was performed.