The VVBlue assay: a plate-readable, dye exclusion-based cell viability assay for the toxicological testing of chemicals†

A viability test for in vitro cultures, based on the intake of the textile dye alphazurine A by dead cells and its exclusion by viable cells, is described. This test uses the affinity of alphazurine A for proteins, so that the dye is retained in dead cells even after rinsing, while its anionic character prevents it from entering live cells. This feature makes this dye exclusion test amenable to a reading in a plate format. The alphazurine viability test provides an indicator of the absolute number of dead cells present in the culture well. To reach a cell viability index, a “dead cells” control (e.g. cells killed with ethanol) must be added. We also describe a double viability test, which first uses the alphazurine assay to provide the number of dead cells then a crystal violet assay to provide an index of the number of cells present in the plate. This double test provides a complete appraisal of the situation in the cell culture wells, and has been compared to other viability tests such as propidium iodide exclusion or tetrazolium reduction. Its performances to study the toxicity of substances such as pigments are also established, and allowed us to publish the first public toxicological data on the recently described Pigment Blue 86.