Quantitation of dye binding by cell monolayers in a microtiter system

Summary A semi-automated system has been developed for the quantitation of dye binding to cultured eukaryotic cells. It is based on staining precisely controlled numbers of cells seeded into microtiter trays. Cell-bound stain is then released using an appropriate solvent and quantitatedin situ by measuring absorbance in a single beam ELISA reader with an interactive microcomputer link. In order to illustrate potential applications of this approach, the time course of dye—monolayer association and influence of cell number and stain concentration on staining has been examined for four dyes, Crystal Violet, Naphthol Yellow S, Ethyl Green and Pyronin Y. In addition, the effect of sequential and simultaneous staining was examined for Ethyl Green and Pyronin Y. The results provide evidence for the overall reliability of this approach as well as revealing several interesting features in the individual procedures examined. The combination of microtiter technology and computer link make the system particularly well suited to the efficient investigation of the permutations involved in optimizing conditions for a given staining procedure, as well as analysis of the thermodynamics of dye substrate interaction. Overall, the approach is viewed as an intermediate between artificial gel systems and microdensitometry.