Synthetic aβ1 42 peptide

sAβ_1–42 was prepared from synthetic Aβ_1–42 peptide (Bachem) as previously described [27 , 50 (link), 51 (link)]. Briefly, 1 mM HFIP (1,1,1,3,3,3-hexafluoro-2-propanol, Sigma) was added to dissolve synthetic Aβ_1–42 peptide and incubated at room temperature (RT) for 1 h. Then, HFIP was evaporated and dried from aliquots to make peptide film. Peptide film was dissolved in 1 mM dimethyl sulfoxide (DMSO) and Ham’s F-12 (phenol red-free, ThermoFisher scientific) was added for dilution and incubated over 12 h at 4 °C for oligomerization. sAβ_1–42 oligomer was confirmed by western-blot before experiments. Unless otherwise indicated, prepared sAβ_1–42 was diluted with cultured neurobasal media to the final concentration of 200 nM and treated to neurons for 2 h. To eliminate the sAβ_1–42 effects, diluted sAβ_1–42 was preincubated with Aβ antibody, 6E10 (Covance) for 2 h before treatment.

Aβ1-42 fibrils were obtained using literature procedures described earlier [26 (link)]. Briefly, synthetic Aβ1-42 peptide (1 mg) (Bachem, Torrance, CA) was initially dissolved in DMSO (50 μL) and diluted with the addition of mQ-H₂O (925 μL). Finally, 1 M Tris-HCl (25 μL; pH 7.6) was added to the peptide solution to obtain a final peptide concentration (222 μM; 1 mg/mL) [27 (link)]. Thereafter, the peptide solution was incubated for 30 h at 37°C with shaking at 1,000 rpm in an Eppendorf Thermomixer. The fibril formation was confirmed by ThioT fluorescence. For determining the concentration of fibrils, the fibril reaction mix was centrifuged at 15,000×g for 15 min to separate the fibrils from the monomer. The concentration of Aβ monomer in the supernatant was determined in a BCA protein assay.