Nucleoview software

NRK-52E cells (1 × 10⁵ cells/well) were pretreated with 80 μg/mL SCAP-ex or 10 μM losartan in 24-well plates for 30 min. Next, 15 μM cisplatin was added and the cells were cultured for another 24 h. The cultured cells were then centrifuged at 1500 rpm for 5 min. The supernatant was removed and the precipitate was suspended in 200 μL of PBS. The suspension was mixed continuously while 800 μL of cold ethanol was added, and the cells were stored at −20 °C overnight. The thiol levels were measured using VitaBright-48 (VB48, ChemoMetec A/S, Lillerod, Denmark) and the number of dead cells was determined using propidium iodide (PI, ChemoMetec A/S, Denmark), according to the instructions provided by the manufacturer of the cell-vitality kit (ChemoMetec A/S, Denmark). The results were obtained using a NucleoCounter NC-250 and the NucleoView software (ChemoMetec A/S, Denmark).

RAW264.7 cells were seeded overnight at a density of 1 × 10⁶ cells/well in a 6‐well plate. The next day, cells were exposed to the indicated concentrations of CuO NPs or CuCl₂ for 6 h. In parallel, cells exposed to etoposide (10 µM) (Sigma) as a positive control. Then, cells were harvested and processed for caspase assay using Vybrant™ FAM Caspase-3 and -7 Assay Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were incubated with FLICA™ reagent and Hoechst in PBS at 37 °C for 1 h, protected from light. After incubation, the cells were washed twice in 1X apoptosis wash buffer. Next, cells were resuspended in apoptosis wash buffer supplemented with propidium iodide (PI) provided with the kit and immediately analyzed using the NucleoCounter^® NC-3000™ (ChemoMetec, Allerød, Denmark). The data were analyzed and plotted using NucleoView™ software (ChemoMetec).