In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues

Méry, Benoite; Guy, Jean-Baptiste; Vallard, Alexis; Espenel, Sophie; Ardail, Dominique; Rodriguez-Lafrasse, Claire; Rancoule, Chloé; Magné, Nicolas

Adenosine triphosphate is produced by living cells and is a sine qua non condition to cellular life. During the cell death process, the potential to synthesize ATP decreases, whereas endogenous cytoplasmic adenosine triphosphatases remove any remaining ATP. Therefore, cell viability can be measured by counting the ATP amount from cultured cells which is a valid marker of viable cells, under certain experimental conditions. 26 Sensitive quantification of intracellular ATP is achieved by luciferase-based assays which are amenable to HTS studies. It is based on the ability of firefly luciferase to generate a luminescent signal. Hall et al developed mutant forms of luciferase, which were stable to environmental extremes as well as homogeneous reagent for measuring ATP directly from cultured cells. The luminescent ATP detection provides many advantages and has become the technique of choice for measuring cell viability in HTS laboratories: it is both easy and non time-consuming, whereas test compounds do not interfere with fluorescent assay methods. Moreover, the ATP assay is the most sensitive microplate assay available for detecting viable cells in culture as less than 10 cells per well can be labeled. 27, 28 However, it should be recognized that decreased intracellular ATP concentrations may result from nonlethal perturbations, including cessation of proliferation (senescence or contact inhibition, for instance) and inhibited mitochondrial respiration. Thus, the measurement of ATP is not always directly linked with cell viability. 25 Alternatively, the measurement of metabolic activity can be achieved using tetrazolium salt as dead cells cannot metabolize the substance. The incubation of viable cells with tetrazolium salt leads to the production of colored formazan product, associated with metabolic activity. Subsequently, colorimetric assays MTT, XTT, or WST-1 will be used to measure cell survival. Unfortunately, similar to the ATP-based assays, several factors may inhibit mitochondrial reductases, and consequently, the production of colored formazan product may not provide unequivocal data on cell viability. 29 Finally, as ATP-based and MTT-based assays are highly susceptible to metabolic interferences and may generate false-positive results, their results must be validated in a secondary time with cell death markers. Metabolism-oriented tests remain useful to get preliminary information but cannot identify cell death modes and discriminate between cytotoxic and antiproliferative effects.