Quantification of adherent and nonadherent cells cultured in 96-well plates using the supravital stain neutral red

In this study we present a rapid, simple, sensitive, inexpensive, and environment-friendly assay for determination of the number of adherent or nonadherent cells cultured in 96-well plates using the supravital stain neutral red. We describe a validation of the method and demonstrate its application to study the effects of hormones (i.e., parathyroid hormone) and cytokines (i.e., tumor necrosis factor-alpha) on the growth of primary cultures of adherent osteoblast-like cells. In addition we show that this method can also be applied to conveniently determine proliferation of cells which grow in suspension, like the CTLL-2 and B-9 cells, which are widely used to measure IL-2 and IL-6 bioactivity, respectively. In these bioassays the changes in optical density induced by IL-2 and IL-6 measured with the neutral red assay are directly comparable with the relative changes measured with the [3H]thymidine incorporation assay. For all types of cells tested, the optical density at 550 nm was directly proportional to the number of cells. The assay, which can be used for different purposes, is an excellent alternative to already existing methods. It is not only easy to perform but also very reproducible, making it ideal for screening of large numbers of samples. Therefore this assay offers a reliable and flexible tool to determine both stimulatory and inhibitory effects of hormones, cytokines, and drugs on cell growth or to study the effects on cell viability.