APPLICATION AND EVALUATION OF THE ALAMARBLUE ASSAY FOR CELL-GROWTH AND SURVIVAL OF FIBROBLASTS

Cell proliferation assays are essential to developing an understanding of the molecular mechanisms that modulate cell growth and differentia tion. In this paper, we describe the application of alamarBlue, a new and versatile metabolic dye, for the detection of Swiss 3T3 fibroblast proliferation and/or survival. As a redox indicator, alamarBlue is re duced by reactions innate to cellular metabolism and, therefore, provi des an indirect measure of viable cell number. Various assay parameter s were optimized for a 96-well format to achieve a detectable range of fibroblast cell number from 100 to 20 000 cells/well, which is simila r to that obtained with traditional (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) and [H-3]thymidine assay techniques . Standard (reference) curves generated with a known fibroblast stimul ator were used to facilitate quantitation and comparison of unknown te st substances. The alamarBlue assay offers the advantages of technical simplicity, freedom from radioisotopes, versatility in detection, no extraction, and excellent reproducibility and sensitivity. We anticipa te that this simple and versatile alamarBlue assay, when used alone or in conjunction with other bioassays, will be a useful tool for invest igating the complex mechanisms of cellular proliferation.