75 cm2 cell culture flask

The antiproliferative effect of Fe₃O₄ MNPs on various cancer cell lines and a normal Chang liver cell line was quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) kit (Sigma-Aldrich) according to the standard method.28 (link) Briefly, cells were allowed to grow in a 75 cm² cell culture flask (TPP Techno Plastic Products, Trasadingen, Switzerland) until 95% confluent. The cells were then seeded into each well of a 96-well microculture plate (TPP Techno Plastic Products) at a concentration of 1×10⁵ cells/mL and treated with Fe₃O₄ MNPs at various concentrations. After incubation for 72 hours at 37°C in a 5% CO₂ incubator (Binder GmbH), 25 μL of a 5.5 mg/mL MTT solution was added to each well, covered with aluminum foil, and incubated for a further 3 hours in the dark. Immediately afterwards, the medium was aspirated and the remaining purple formazan was lysed with MTT solution. The assay was performed in triplicate. The optical density was then measured at 570 nm using an enzyme-linked immunosorbent assay universal microplate reader (Bio Tek Instruments, Inc., VT, USA). The inhibitory concentration 50 (IC₅₀) value was determined from the absorbance versus concentration curve. Dimethyl sulfoxide 0.1% was used as the negative control.