*4.2. Cell Culture*

Colon cancer cell line (HT-29), breast cancer cell line (MCF-7), cervical cancer cell line (HeLa), and renal epithelial cell line (293T) were cultured in DMEM medium supplemented with 20% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics. The cells were incubated in a 5% CO2 incubator at 37 ◦C.

## *4.3. MTT Assay for Cell Viability*

The cytotoxicity of fucoidan toward HT-29, Hela, MCF-7, and 293T cells was determined using the MTT assay. The cells were seeded in a 96-well plate (6 × 10<sup>3</sup> cells/well), and fucoidan (0, 100, 200, 400, and 800 μg/mL) was added after 24 h, Doxorubicin, 5 μg/mL, was used as a positive control. After incubation for 48 h, a 10 μL aliquot of MTT solution (5 mg/mL) was added to each well, and the cells were incubated for an additional 4 h. The medium was then aspirated, 100 μL of DMSO was added to each well, and the plates were vortexed for 3 min until thoroughly mixed. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader (Sunrise, Tecan, Austria).
