Exoquick tc ultra ev isolation kit for tissue culture media

According to the manufacturer’s indications, exosomes delivered to culture media were purified with ExoQuick TC® ULTRA EV Isolation Kit for Tissue Culture Media (System Biosciences, Palo Alto, USA), and a fraction was used to assess purification by Western blot using the exosome markers CD81, CD9, and CD63 (EXO AB kit, System Biosciences) (Supp. Fig. 1). Western blot was performed as described previously [32 (link)]. Cells were harvested after the indicated incubation times and centrifuged (400 g) for 5 min. Cell pellets were stored for later processing, and the cell culture media supernatant was collected and stored at − 80 °C until processing. Five milliliters of media were used to purify exosomes. Exosome purification was assessed by Western blot using exosome antibodies provided by System Biosciences (Palo Alto, USA). As a control, media containing charcoal-stripped FBS were purified in parallel.

N2a-APPswe cells were transfected with a non-silencing control siRNA or active siRNA against Prnp (Prnp3 plus Prnp4) and/or transfected with a pCB6 expression vector containing either PrP^A116V or WT mouse PrP. After incubation for 24 h, siRNA and the transfection reagent were removed and replaced by OPTI-MEM I (Invitrogen) for 24 h incubation. Exosomes were prepared using ExoQuick-TC ULTRA EV Isolation Kit for Tissue Culture Media (System Biosciences, SBI, Palo Alto, CA), following the manufacturer’s instructions^{26 (link),27 (link)}. Briefly, cell culture media was collected and centrifuged at 3,000 × g for 15 min to remove debris. The supernatant was mixed with ExoQuick-TC and incubated at 4 °C overnight. The mixture was centrifuged at 3,000 × g for 10 min. The pellet was resuspended in Buffer A and passed through the purification column. The purified exosomes were then prepared for transmission electron microscopy (TEM) by negative staining, and for Western blotting to assess Aβ, PrP, APP, and exosome markers Alix, flotillin, and CD-63 proteins.