Spectrophotometer plate reader

DNA synthesis was assessed by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into cellular DNA. The test was performed after 72, 120 and 168 h of culture. The assay was performed using BrdU Cell Proliferation ELISA Kit (Abcam, Cambridge, UK) in accordance with the manufacturer’s instructions. After incubation with BrdU overnight at a temperature of 37 °C, cells were fixed and DNA was denaturated. Incorporation of BrdU was determined by incubation with anti-BrdU monoclonal antibody. Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) was used as a secondary antibody. Signal intensity was measured with a spectrophotometer plate reader (BMG Labtech, Ortenberg, Germany) at a wavelength of 450/550 nm.

DNA synthesis was assessed by measuring the incorporation of 5‐bromo‐2‐deoxyuridine (BrdU) into cellular DNA. The test was performed after 48, 96 and 168 hrs of culture. The assay was performed with BrdU Cell Proliferation ELISA Kit (Abcam, Milton, Cambridge CB4 0FL, United Kingdom) in accordance to manufacturer's instruction. Briefly, cells were incubated with BrdU overnight at 37°C. Next, cells were fixed and DNA was denaturated. Incorporation of BrdU was determined by incubation with anti‐BrdU monoclonal antibody. Goat antimouse IgG conjugated with horseradish peroxidase (HRP), was used as a secondary antibody. Colour reaction was developed using 3,3,5,5‐tetramethylbenzidine (TMB). Signal intensity was measured with a spectrophotometer plate reader (BMG Labtech, Ortenberg, Germany), at a wavelength of 450/550 nm.

Human breast cancer cell line (MDA-MB 231) which was obtained from American Type Culture Collection (ATCC), seeded (5 × 10³ cells, passage no. 41) in 96-well cell culture plates using DMEM medium with 10% FBS as the culture medium, and incubated in 5% CO₂ incubator at 37 °C. After 24 h, the attached cancer cells were treated with serial concentrations of CD NPs, ZD NPs, CD, ZD, and standard chemotherapy (fluorouracil) as well as DE, copper chloride and zinc chloride. Following 48 h in 5% CO₂ incubator, MTT (5 mg/mL) was incubated with the untreated and treated cells for 4 h then DMSO was added and the absorbance was read at 590 nm^{43 (link)} using spectrophotometer plate reader (BMG LabTech, Germany). The IC₅₀ value, at which 50% cancer growth inhibition, was estimated by GraphPad Prism 9. Additionally, a change in the morphology of the treated breast cancer cell was observed using phase contrast inverted microscope with a digital camera (Olympus, Japan).