Hilyte fluor 555 labeled

25‐hydroxycholesterol, bovine serum albumin (BSA), and trypan blue were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Aβ protein (human, 1–42; Aβ‐42) was obtained from Peptide Institute Inc. (Osaka, Japan), HiLyte FluorTM 488‐labeled (λex = 503 nm, λem = 528 nm) Aβ‐42 and HiLyte Fluor‐555‐labeled (λex = 551 nm, λem = 567 nm) Aβ‐42 were obtained from Anaspec, Inc. (Fremont, CA, USA). ER‐Tracker Blue‐White DPX, Oregon Green 488 taxol, Roswell Park Memorial Institute 1640 (RPMI1640) medium, FBS, and Alexa Fluor 555‐conjugated cholera toxin B subunit (CT‐B; λex = 555 nm, λem = 565 nm) were obtained from Invitrogen (Eugene, OR, USA). Phosphate buffer salts (PBS), and Tris (hydroxymethyl) aminomethane (Tris) were purchased from Takara Bio Inc. (Shiga, Japan) and Kanto‐Chemical (Tokyo, Japan), respectively.

Beta-amyloid (1–42) and HiLyte Fluor 555-labeled and non-labeled human Aβ₁₋₄₂ (1 mg) peptide were purchased from AnaSpec and Aladdin, UK, respectively. Prior to use, 1 mg freeze-dried powder of Aβ₁₋₄₂ and hexafluoroisopropanol (HFIP) were placed on ice for pre-cooling. Two hundred and twenty-two microliters of HFIP was injected into the reagent bottle, sealed and mixed gently, and kept at room temperature for 60 min until the liquid became clear to obtain the Aβ-HFIP solution (1 mM). Four sterile 1.5-ml EP tubes were taken, and the Aβ-HFIP solutions, divided into four equal parts (55 μL each), were individually placed in each tube. HFIP was dried by a vacuum freeze-drying apparatus, and an Aβ peptide film was obtained, which was then stored at −20°C. In a separate tube, 11 μL DMSO was added to the Aβ peptide membrane. After 10 min of water bath ultrasound (power 300 W, frequency 35 Hz), Aβ-DMSO solution (5 mM) was obtained. The pre-cooled 539 μL PBS solution (100 μM) was added to Aβ-DMSO solution and mixed gently. To further promote the formation of the fibrils, the solution was incubated at 37°C for 1 week. Prior to use, it was diluted 100 times to 1μM in the medium and then cultured with cells.