Adherence Assay for Anti-Cancer Drugs

Cells were exposed for 72 h to the mono drugs (1 μg/mL Tz, 1μg/mL Pz, 1 μg/mL T-DM1, 0.1 μg/mL Lp) and the combinations (1:0.1 μg/mL Tz + Lp, 1:0.1 μg/mL T-DM1 + Lp, 1:1 μg/mL Tz + Pz) with and without the transfection with specific siRNAs versus FAK, paxillin, and cortactin. After the treatments, cells were trypsinized and suspended in PBS containing trypan blue to determine the number of viable cells and seed the same viable cell density for each experimental condition. Briefly, 3 × 10⁴ SKBR3, 5 × 10⁴ BT-474, and 3 × 10⁴ SKBR3-RTz cells/well were seeded into 96-well plates previously coated with 1% sterile gelatin (Sigma-Aldrich). Cells were incubated at 37 °C for 2 h. Non-adherent cells were removed by gentle washing with PBS. The attached cells were fixed with 4% p-formaldehyde and stained with 10% ethanol/crystal violet for 20 min. Absorbance at 570 nm was measured using a microplate reader (MULTISKAN EX; Thermo Scientific, St. Leon-Rot, Germany). The absorbance is directly proportional to the number of adherent cells. The results are expressed as the percentage of cell adhesion, normalized with respect to control, which is considered 100%. Images were captured using a Nikon Eclipse E200 microscope coupled with a high-resolution CCD digital camera. The protocol was previously described in Castro-Guijarro et al. (2022) [36 (link)].