Black walled optically clear bottomed 96 well plates

Nrf2 expression was detected by immunofluorescence assay. H9C2 cells were cultured in black-walled optically clear-bottomed 96-well plates (Corning Life Sciences, Acton, MA, USA). After the treatment, the cells were incubated with anti-Nrf2 antibodies overnight at 4°C. The antibodies were washed for three 10-minute periods with PBS. The cells were then incubated for 1 h in fluorescein-conjugated secondary antibodies. Subsequently, cell nuclei were stained with DAPI (1 μg/ml for 10 min). The stained cells were washed with PBS and visualized using a fluorescence microscope at 200x magnification.

Human neuroblastoma SH-SY5Y cells, which overexpressing the Swedish mutant form of human APP (referred to as “APPsw cells”) as an AD cell model by using copper to trigger the neurotoxicity of Aβ, were used to evaluate the effect of MSL. In brief, SH-SY5Y cells were cultivated in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and maintained under standard conditions of 37°C, 5% CO₂, and 95% humidity. For immunofluorescence analysis, cells were seeded at a density of 8000 cells per well in black-walled optically clear-bottomed 96-well plates (Corning Life Sciences, Acton, MA, USA). Cells were treated with MSL (50 μM, 100 μM and 200 μM) and copper (250 μM) and incubated for 12 h. Then, cells were collected to evaluate the effects of  MSL.