Bca assay

Murine neuroblastoma Neuro2a (N2a) cells, human APP751-stable transfected N2a cells, and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. One day before EV isolation, the cells were transferred to a serum-free medium. EVs were collected from cell supernatants following differential ultracentrifugation. The supernatants were sequentially centrifuged at 2000 × g for 10 min, 10,000 × g for 30 min to remove cells and debris, and then 100,000 × g for 1 h to pellet the EVs. EV protein content was measured with the BCA assay (Nacalai Tesque, Kyoto, Japan). Sizes and densities of the EVs suspended in PBS were analyzed with a qNano system (Izon Science, Cambridge, MA) using NP200 nanopores and qNano Izon analysis software. CPC100 was used as the calibration sample in this study. Microphotographs of the EVs were obtained with an HD-2000 scanning transmission electron microscope (Hitachi, Tokyo, Japan).

Bt-labeled BSA (Bt-BSA) was prepared by incubating 5 mg/ml BSA (Iwai Chemicals) with a 10-fold molar Bt-PE-maleimide (Dojindo Laboratories) in PBS at 25 °C for 16 h. After incubation, the aliquots were dialyzed against PBS. Biotin-labeled Plg (Bt-Plg) was prepared with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) following the manufacturer’s instructions. The AGEs were prepared according to a previous report^{9 (link)} by incubating 1.0 mg/ml BSA with 25 mM DHA (Sigma) in PBS at 37 °C under atmospheric oxygen. After 72 h, the reactants were collected and dialyzed with PBS. For the preparation of Bt-labeled acylated proteins (Ac-BSA, Sc-BSA, and Ma-BSA), 5 mg/ml Bt-BSA was added to equal volume of a saturated solution of sodium acetate with continuous stirring on ice, followed by the incubation at 4 °C for 1 h with 2 mM acetic anhydride, succinic anhydride, or maleic anhydride, respectively. After incubation, the aliquots were dialyzed against PBS. The protein concentrations were measured by a BCA assay (Nacalai Tesque Inc.). DHA-modified Bt-labeled N-pentylamine (DHA-Bt-PA) was prepared upon incubation of 6 mM EZ-Link pentylamine biotin (Thermo Fisher Scientific) with 25 mM DHA at 37 °C for 72 h.

Cells were harvested in 200 μl lysis buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100). An aliquot of the lysate was mixed with the same amount of Folch solution (2:1 v/v chloroform/methanol). The lower phase was collected, and TG content determined, using a Triglyceride Determination Kit (Wako). Another aliquot of the lysate was used for protein determination by BCA assay (Nacalai Tesque).