Beta amyloid 1 42

All the chemicals used in this study are commercially purchased and used without further purification. Lipofectamine (LPS) was purchased from Thermo Fisher (00‐4976‐93); Beta Amyloid (1–42) was procured from Anaspec (AS24224), Mammalian protein extraction reagent (MPER; #78 505)), EDTA, Protease inhibitor (78 425) were purchased from Thermo Fischer Scientific. For 2D cell culture, murine microglia BV2 cell was used, human microglia HMC3 and cultured in Dulbecco's modified Eagle's media (12‐707F; Lonza) supplemented with 10% fetal bovine serum (FBS), L‐Glutamine (25‐005‐Cl; Corning), and Penicillin/streptomycin (DE17‐602E; Lonza). Cells were cultured in a 37 °C incubator with water‐saturated air and a 5% CO₂ atmosphere and splitting was done every 2–3 days using trypsin (BE17‐161E; Lonza).

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.