T 75 flasks

BT474 cells (ATCC HTB-20) were maintained in a suspension in Hybri-Care medium (ATCC 46-X) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in T-75 flasks (CELLTREAT). T47D cells (ATCC HTB-133) were maintained in a suspension in RPMI-1640 medium (ATCC 30-2001) containing FBS, 4 mg/L insulin, 100 U/mL penicillin, and 100 μg/mL streptomycin in T-75 flasks (CELLTREAT). For the cytotoxicity assays, cells were planted into 96-well plates with 10,000 cells per well. These plates were incubated overnight at 37 °C and 5% CO₂. Serial threefold dilution was applied to the ADC samples with the corresponding medium from 5000 to 0.085 ng/mL. The samples were added to three wells (150 μL per well) with every single concentration, and the cells were cultured at 37 °C and 5% CO₂ for three days before the addition of cell counting kit-8 (Sigma). The absorbance of formazan released by viable cells was measured at 450 nm using a spectrophotometer after incubation for 2–3 h at 37 °C and 5% CO₂. Finally, the EC₅₀ values and the cell viability curve were calculated using GraphPad Prism software.