3.5. Cytotoxicity Assay

The MDA-MB-231 and HT-29 cell lines were seeded at a density of 5000 cells/well in triplicates on a 96-well tissue culture plate (Corning, Sigma-Aldrich Co., St. Louis, MO, USA). The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] test was used to determine the cytotoxicity of the S2 magnetic nanoparticle treated with an AMF with a frequency of 384.50 kHz and a 350 G field strength. The control group (C) cells were maintained at 37 °C in the incubator throughout the experiment. One set of cells (F) was treated with a magnetic field without nanoparticles to examine the effect of the magnetic field. One more group of cells (P) was treated with nanoparticles without a field to study their effect alone. For the AMF treatment, the cells (P + F) were treated with 1 mg/mL of S2 nanoparticles. The cell viability was measured at 0 and 24 h post-AMF treatment. At specified time intervals, the cells were washed three times with phosphate-buffered saline (PBS) to remove the nanoparticles from the cell surface, followed by the addition of 100 µL of culture medium and 10 µL of MTT solution (5 mg/mL in PBS) and incubation for 2 h under humidified air and 5% CO₂ at 37 °C. The MTT-containing medium was then removed, and formazan crystals were dissolved in 100 µL of dimethyl sulfoxide. The absorbance of the MTT assay was measured at 570 nm using an ELISA plate reader Synergy^TM HTX microplate reader (BioTek, Winooski, VT, USA). The cell viability percentage was calculated by using the following equation: