Metabolic and DNA-based assays provide equally reliable measures of cell viability across experimental conditions

Accurately and reliably assessing cell viability is a fundamental necessity in biomedical research. However, many cytotoxic compounds used in cancer research have both on- and off-target effects on cellular metabolism, which may confound metabolism-based cell viability assessments, whereas DNA- and protein–stain-based methods are thought to be less susceptible to this limitation. This study aimed to compare the performance of the metabolic assay CellTiter-Blue (CTB) with the DNA-staining assay Crystal Violet (CV) after treatment with cytotoxic drugs with and without off-target effects, mitochondria-inhibiting compounds, and diaphorase inhibitors in three esophageal adenocarcinoma cell lines, as a formal comparison of the two methods has not yet been established. CTB and CV returned highly comparable half-maximal inhibitory concentrations at 72 h, with strong correlations between readouts for nearly all tested compounds. Gene and protein expression analysis of cell lines with differential CTB reduction capacity identified NQO1 as the diaphorase enzyme responsible for the CTB reaction. CTB underestimated cell viability by up to 53% compared with CV after short-term pharmacological diaphorase inhibition, but there was no difference for any of the other tested compounds in this timeframe. This was confirmed functionally by an shRNA-mediated knockdown of NQO1, resulting in a significantly reduced capacity to metabolize CTB without compensatory changes in the expression of other diaphorases. As CTB and CV results were comparable at 72 h after treatment with NQO1 inhibitors, we conclude that CTB is a robust method across nearly all inhibitors and in most realistically applied timeframes.