Solubilization solution

HT 22 cell was purchased from the Korean Cell line Bank (Seoul National University, Republic of South Korea) and cultured in the Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, USA) with 10% foetal bovine serum and 1% penicillin. The cytotoxic effect of synthesized Aβ_1-42, Aβ_4-42, and Aβ_pE3-42 was investigated using the previously reported MTT assay protocol^{24 (link)}. Cultured HT22 cell (8 × 10³ cells/well) was seeded on a 96-well plate. Each Aβ variant (10 μM) was prepared in starvation medium (0.5% foetal bovine serum and 1% penicillin in DMEM) and treated on the plate for 24 h at 37  ${}^{\circ }$ C. 15 μL of MTT reagent (Promega, USA) was added to each well and incubated for additional 4 h at 37  ${}^{\circ }$ C. Finally, 100 μl of solubilization solution (Promega, USA) was added for 30 min at 37  ${}^{\circ }$ C. The absorbance was measured at 570 nm.

After 24 h of incubation with the
sample of the protein aggregates, the cells were stained using CellTiter
96 nonradioactive cell proliferation assay (MTT) kit (Promega, Madison,
Wisconsin). Cells were incubated with the dye for 4 h at 37 °C
under 5% CO₂. Next, solubilization solution (Promega, Madison,
Wisconsin) was added and incubated for 1 h at 37 °C under 5%
CO₂. Absorption measurements were made in a plate reader
(Tecan, Männedorf, Switzerland) at 570 nm. Every well was measured
25 times in different locations.

Cells were seeded at 1 × 10⁵/well and treated with inhibitors for 48 hours. 15 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; 5 mg/ml; Sigma) was added to each well and incubated at 37°C for 4 hours. 100 μl solubilization solution (Promega) was added to each well, and cells were incubated over night at 37°C. Absorbance was measured by spectrophotometry at wavelengths 590 and 630 nm.