4.8. In Vitro Cytotoxicity Assays

In vitro cytotoxicity of conjugates was assessed on several antigen-positive cell lines. Cells were plated in 96-well plates at an appropriate density depending on the cell line (between 1000 and 10,000 cells per well in 100 µL of appropriate culture media) and incubated at 37 °C for 24 h. Serial dilutions of the tested compound previously dissolved in culture media were added, and incubation was carried out at 37 °C for 144 h. MTT (5 mg/mL, 20 μL, Sigma-Aldrich) was added into the wells, and incubation was continued for 2 to 4 h at 37 °C. Culture media was then carefully removed, and well content was homogeneously dissolved with 0.1 N HCl/isopropanol. Absorbance values were measured on a Thermo Scientific Multiskan EX microplate reader using a wavelength of 570 nm (with a reference wavelength of 690 nm). The IC₅₀ concentration values compared to untreated control cells were determined using inhibition dose response curve fitting (GraphPad Prism 9).