*3.12. Evaluation of Cytotoxic Activity*

The cytotoxic activity of the fucoidan extracts was measured using the MTT assay. Briefly, cells were cultured in growth medium with 37 ◦C humidified 5% CO2 atmosphere for 24 h. All fucoidan extracts were prepared as a 20 mg/mL stock solution by thoroughly dissolving fucoidan powder in phosphate-buffered saline (PBS). The medium was then removed, and the cells were treated with various concentrations of fucoidan extracts. Final concentrations of 0, 50, 100, 200, 300, 400, and 500 μg/mL were obtained by diluting the stock solution with serum-free medium to prevent the fucoidan extract from potentially losing its potency in the presence of serum. After 48 h of treatment, cells were washed with PBS once, and MTT stock solution was added to each culture so that the final concentration of MTT in the medium was 0.1 mg/mL. After 2–4 h of incubation, the formazan was solubilized by adding isopropanol and measured by absorption at 560 nm. The cell viability was expressed as a percentage of MTT reduction.
