A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.

There is currently much interest in the role of mediators that regulate cell proliferation. Methods to assay proliferative effects of such mediators usually involve cell counting techniques, which are tedious to perform, or methods based on uptake of radiolabelled thymidine, which may be prone to errors caused by precursor pool artefacts. We describe here an assay for estimating the number of adherent cells present in a microculture and its application to the study of growth factors. The assay depends on the binding of Methylene Blue to the fixed monolayer at pH 8.5 and, after washing the monolayer, release of dye by lowering pH. The use of an elution solvent containing acidified ethanol ensures a linear correlation between absorbance of the dye and cell number, and enables the assay to be carried out in 96-well plates measuring absorbance with an automated vertical light-path microplate photometer. The assay is rapid, highly reproducible and easy to perform, making it ideal for screening large numbers of samples. It was shown to be applicable to a number of foetal and adult cell lines derived from man and experimental animals. It was also demonstrated to be useful for assaying purified growth factors and detecting growth promoting activity in cell and tissue extracts.