2.5. Cell Adhesion Assay

In order to evaluate the adhesion potential of MDA-MB-231 breast cancer cells, the following adhesion protocol was conducted, as it was described by previous works [39,40]. Briefly, 96-well plate was coated with collagen type I solution (40 μg/mL) and kept at 4 °C. After 12 h, the solution was removed, and the plate was air-dried; 3% BSA in PBS solution was added in each well, for 30 min, to block non-specific adsorption. Then the solution was removed, and the plate was washed with PBS and air-dried. Cells treated for 24 h prior to the adhesion assay were detached with PBS-EDTA 1× and resuspended in serum-free medium with 0.1% BSA. and seeded at a density of 2 × 10⁴ cells/well. Cells were incubated for 30 min, to allow adhesion to the surface. Non-adherent cells were removed with serum free medium, and then cells were incubated with medium supplemented with 10% FBS for 2–3 h for recovery. After the incubation period, Premix WST-1 (water-soluble tetra-zolium salt) Cell Proliferation Assay System (Takara Bio Inc., Göteborg, Sweden) was added at a ratio 1:10, and the absorbance at 450 nm was measured (reference wavelength at 650 nm).