To investigate the cytotoxicity of the unconjugated small-molecule TOP1 inhibitors and ADCs, respective cells were incubated for 4 days (small molecules) and 7 days (ADCs) with increasing concentrations of small molecules (0.015-1,000 nmol/L) and ADCs (0.05-3 ug/mL) to generate a dose-response curve. Killing was analyzed using resazurin cell viability dye at a final concentration of 55 umol/L (Merck) by dividing the fluorescence from control cells in medium by the fluorescence of ADC-treated cells. Fluorescence emission at 590 nmol/L was measured on a microplate reader Infinite 200 Pro (Tecan Group Ltd.).