Quantitative determination of apoptotic death in cultured human pancreatic cancer cells by propidium iodide and digitonin

Abstract

We present here the efficacy of an in vitro cytotoxicity assay which can measure rapidly both apoptotic dead cells and cell growth rate, quantitatively. Using a multi-well plate reader, the fluorescence intensity of propidium iodide (PI) corresponding to dead cells and to total cells after digitonin treatment were measured in cultured human pancreatic cancer cells following exposure to etoposide. The percentage of dead cells measured by this assay was well correlated to that determined by Trypan Blue staining. Furthermore, the cell growth rate determined simultaneously was also correlated to the cell number counted directly using a microscope. We demonstrate that this method, which was originally established for evaluating necrosis, could be applied to measure apoptotic cell death. Taken together, this simple assay is useful for testing the efficacy of anti-cancer agents and for investigating the molecular mechanisms of apoptosis in the cultured cells.