Simultaneous Evaluation of Cell Viability by Neutral Red, MTT and Crystal Violet Staining Assays of the Same Cells

By combining three separate toxic assays, the neutral red (NR), MTT and crystal violet staining assays (CVS), we developed a convenient assay method, the NMC assay, in which the NR, MTT and CVS assays are performed consecutively on the same cultured HeLa cells. The NMC assay is performed as follows: the cultured HeLa cells are first treated with NR, and thereafter with MTT; the NR extracted with 30% methanol and the MTT extracted with isopropanol are measured spectrophotometrically (NMC-NR and NMC-MTT assays); the residual cells adhered on the microplate are then stained with crystal violet (NMC-CVS assay). The absorbance values in each of the NMC assay correlated with the viable cell count, as when the assays were performed individually. The EC(50) values of sodium lauryl sulfate (SLS) obtained by the NMC-NR, NMC-MTT and NMC-CVS assays in the HeLa cells were also in agreement with that obtained by each individual assay. Some chemicals, namely monensin, diisopropanol amine and antimycin A, gave different dose-cell viability curves in the NMC-NR, NMC-MTT and NMC-CVS assays depending on their specific mechanisms of action.