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

TLDR

Cell proliferation is regulated by various mediators, but existing assays are tedious or error‑prone, prompting interest in more efficient methods. The authors developed an assay to estimate adherent cell numbers in microculture and to evaluate growth factor effects. The assay uses methylene blue binding to fixed cells at pH 8.5, followed by dye release at lower pH and acidified ethanol elution, yielding a linear absorbance–cell number relationship measured in 96‑well plates with an automated photometer. The method is rapid, reproducible, and easy to perform, enabling screening of many samples, and it works with various fetal and adult cell lines and for detecting growth‑promoting activity in purified factors and extracts.

Abstract

ABSTRACT 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.