2.8.2. Hemolysis

The hemolysis assay was carried out according to ISO 10993-4 [15]. Briefly, 270 µL of erythrocytes were placed in a 96-well plate, and treatment with CuONPs was used at different concentrations (33.3, 35.52, 37.74, 39.96, and 42.18 µg/mL). The final volume was completed to 300 µL with DMEM medium, no treatment was added to the negative control, and 30 µL of Triton-100 at 20% was used for the positive control. The plate was left to incubate for one hour at 37 °C and 5% of CO₂. Afterward, the plate was centrifuged for 5 min at 3000 rpm, the resulting supernatant was transferred to another 96-well microplate, and the absorbance at 541 nm was measured in a UV-visible spectrophotometer (Thermo Scientific Multiskan GO, Vantaa, Finland).