Matrigel coated 96 well black plates

DCFH-DA (#S0033, Beyotime, Shanghai, China) was used to detect intracellular ROS levels. Human NPCs were plated in Matrigel-coated 96-well black plates (#3094,Corning, NY, USA) at 2000 cells per well and cultured in medium consisting of 1:1 DMEM/F12: Neurobasal, NEAA, Glutamax, N2 and B27 supplement, 1 ng/mL bFGF. The cells were treated with 10 μM Aβ_1-42 for 24 h and then stained with 10 μM DCFH-DA and 3 μg/mL Hoechst (#C1022, Beyotime, Shanghai, China) for 30 min at 37 °C in a humidified incubator with 5% CO₂. The cells were washed twice with PBS and observed under a laser-scanning confocal microscope (Operetta, Perkin Eimer, MA, USA). Alternatively, the fluorescence was measured by a BioTek SynergyNEO microplate reader (Bio-Tek, Williston, VT, USA) at a 488 nm excitation wavelength and a 525 nm emission wavelength.

JC-1 staining kits (Beyotime, C2006) were used to assess the MMP level of the cells, according to the manufacture’s protocols. Briefly, NPCs were plated in Matrigel-coated 96-well black plates (#3094,Corning, NY, USA) at 2000 cells per well and cultured in medium consisting of 1:1 DMEM/F12: Neurobasal, NEAA, Glutamax, N2 and B27 supplement, 1 ng/mL bFGF. After 24 h of treatment with 10 μM Aβ_1-42, 50 μl of JC-1 staining solution was added to each well and incubated at 37 °C for 20 min. These cells were then washed twice with the staining buffer and observed under a Zeiss Observer Z1 microscope. The fluorescence intensity was detected using the BioTek SynergyNEO (BioTek, Williston, VT, USA) device. The JC-1 monomer was detected at a 490 nm excitation wavelength and a 530 nm emission wavelength. The JC-1 polymers (J-aggregates) were detected at a 525 nm excitation wavelength and a 590 nm emission wavelength. The membrane potential was represented as the ratio of red/green fluorescence intensity.