Cm h2dcfh da

An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (K101-100; BioVision, Milpitas, CA, USA) and CM-H2DCFHDA (C6827; Invitrogen, Waltham, MA, USA) were used for a cell death assay and cellular ROS imaging, respectively. In brief, SH-SY5Y cells (5.0 × 10⁵ cells/well) were seeded on 35-mm glass-bottom dishes (Greiner Bio-One, Kremsmünster, Austria). After being cultured in medium containing 5 μM Aβ₄₂ for 24 h, the cells were washed twice with PBS, and then incubated with annexin V-FITC and propidium iodide (PI) for 5 min at room temperature in the dark. For the detection of ROS, the cells (5.0 × 10⁵ cells/well) were seeded in 35 mm glass-bottom dishes and pretreated with CM-H2DCFHDA (10 μM) for 30 min at 37 °C in the dark, and then treated with 5 μM Aβ₄₂ for 3 h. The cells were washed twice with PBS. Fluorescent images were visualized immediately using a confocal laser scanning microscope (BZ-X700; Keyence, Osaka, Japan).

L6 cells were seeded in 24-well plates at 1 × 10⁵ cells/well for short-term experiments (3-h and 24-h incubation) and at 7 × 10³ cells/well for long-term experiments (10-day continuous exposure). For 3-h exposure, we used 200 µg/mL NPs; for 24-h exposure, we used 10 and 200 µg/mL; and for 10-day exposure, we used 10 and 50 µg/mL NPs. Following incubation, the cells were washed with PBS and incubated with 10 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFH-DA; Molecular Probes, Invitrogen, Carlsbad, CA, USA) in PBS (containing Ca²⁺ and Mg²⁺ ions) for 45 min at 37 °C. Fluorescence intensity was measured with excitation at 492 nm and emission at 527 nm using Tecan Infinite 200 (Tecan, Männedorf, Switzerland). As a positive control, 1 mM H₂O₂ was used. The fluorescence intensity of CM-H₂DCFDA was normalized to the number of cells determined by fluorescence intensity of the Hoechst signal measured with excitation at 350 nm and emission at 461 nm using a Tecan Infinite 200 (Tecan Group Ltd., Männedorf, Switzerland).

ROS levels were determined with the 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate assay (CM-H₂DCFH-DA; Molecular Probes, Invitrogen). After a 24 h incubation with increasing concentrations of NPs, the cells were washed and incubated with 10 μM CM-H₂DCF-DA in DMEM FluoroBrite (Gibco, Thermo Fisher Scientific) at 37 °C for 45 min. We used 0.1 mM H₂O₂ (20 min incubation) as a positive control. The fluorescence was measured using a Tecan Infinite 200 spectrofluorometer (Tecan, Männedorf, Switzerland). The fluorescence intensity of CM-H₂DCFH-DA was normalized to the relative number of cells, as determined by Hoechst 33342. The results are presented as the percentage and standard error of normalized fluorescence intensity compared to the negative control sample for three independent experiments in three replicates.