Effects of serum protein and colloid on the alamarBlue assay in cell cultures

The reagent alamarBlue allows for real-time and repeated monitoring of cell proliferation and cell viability in cytotoxicity assays. Foetal bovine serum (FBS), bovine serum albumin (BSA) and, to a lesser extent, polyvinylpyrrolidone (40,000 mw) produce an apparent decrease in the rate of reduction of the reagent in cell cultures. The effect is attributable in part to a measurement artefact, possibly due to binding of the reduced and oxidized, extracellular forms of alamarBlue to these agents, resulting in absorbance and fluorescence spectral shifts. For dual wavelength spectrophotometric determination, this effect can be corrected using empirical absorbance ratios and applying a general equation of the form: AR (570) = ( A (570) - A (600)R (0)) ( R (RI570) - R (R)R (0)R (R)I570 ) , where AR(570)(0) is the standardized absorbance of the reduced product at zero extracellular protein, A(570) and A(600) are the absorbance at 570 and 600 nm of the culture supernatant, r(0) is the ratio of the absorbance at 570 nm to the absorbance at 600 nm for the oxidized substrate, R(R) is the ratio of the absorbance at 600 nm to the absorbance at 570 nm for the reduced product and R(RI570) is the ratio of the absorbance at 570 nm for the reduced form in the presence of interfering protein to the absorbance at 570 nm in the absence of protein. The factors R(0), R(R) and R(RI570) are determined empirically at defined protein concentrations. After correction of absorbance values, FBS and BSA added to culture medium were found to depress the reduction of alamarBlue in lung fibroblasts and mesothelial cells. The alamarBlue assay is thus sensitive to protein conditions in culture media and assay parameters should be standardized for reproducibility.