Determination of ADC Cytotoxicity in Immortalized Human Cell Lines

Cytotoxicity assays are a necessary first step to triage ADC molecules before moving them forward to relatively time-consuming and expensive in vivo studies. When cells are exposed to ADC molecules, antigen expressing cells can effectively take up those molecules and eventually die as a result of the released payload. This cytotoxic property of ADCs can be evaluated by measuring the percentage of living cells at the end of the incubation period. Tetrazolium colorimetric assay (MTT) is a widely used method that can be used to measure cell viability. Here we describe how to use an MTT assay to measure the cytotoxic effect of ADCs and calculate the corresponding IC₅₀. Besides the cytotoxic behavior on antigen expressing cells, ADCs can also demonstrate bystander killing of antigen negative cells in the vicinity of antigen expressing cells. Here, we report how to use a co-culture experiment to evaluate the bystander effect of ADC with the help of fluorescent protein transfected antigen negative cells.