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Quantitation of dye binding by cell monolayers in a microtiter system

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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.

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References

  • GENTRY, M. K. & DALRYMPLE, J. M. (1980) Quantitative microtiter cytotoxicity assay for Shigella toxin.J. Clin. Microbiol. 12 361–6.

    Google Scholar 

  • GOLDSTEIN, D. J. (1961) Mechanism of differential staining of nucleic acids.Nature (Lond) 191 407–8.

    Google Scholar 

  • GIUGLIANO, L. G., BARER, M. R., MANN, C. F. & DRASAR, B. S. (1984) Tissue culture systems for the examination of bacterial virulence. InModels of anaerobic infection (edited by HILL, M. J.), pp. 189–200. The Hague: Martinus Nijhoff.

    Google Scholar 

  • JAKOBSEN, P., ANDERSEN, A. P., LYON, H. & TREPPENDAHL, S. (1983) Preparation and characterization of Pyronin Y.Microsc. Acta. 87 41–7.

    Google Scholar 

  • JAKOBSEN, P., ANDERSEN, A. P. & LYON, H. (1984) Preparation and characterization of Methyl Green tetrafluoroborate.Histochemistry 81 177–9.

    Google Scholar 

  • LYON, H., JAKOBSEN, P. & ANDERSON, A. P. (1984) Nucleic acid staining with the methyl green-pyronin method comparing the use of pure dyes with commercial dye samples. InAbstracts of the VIIth international congress of histochemistry and cytochemistry (edited by PANULA, P., PAIVARINTA, H. & SOINILA, S.), p. 238, Helsinki.

  • MARSHALL, P. N. & HOROBIN, R. W. (1973) Measurements of the affinities of basic and "mordant" dyes for various tissue substances.Histochemie 36 303–12.

    Google Scholar 

  • PEARSE, A. G. E. (1985)Histochemistry: Theoretical and Applied, Vol. 2, 4th edn. pp. 632–5. Edinburgh, London, Melbourne, New York: Churchill Livingstone.

    Google Scholar 

  • SCOTT, J. E. (1967) On the mechanism of the methyl greenpyronin stain for nucleic acids.Histochemie 9, 30–47.

    Google Scholar 

  • TAS, J., OUD, P. & JAMES, J. (1974) The Naphthol Yellow S stain for proteins tested in a model system of polyacrylamide films and evaluated for practical use in histochemistry.Histochemistry 40 231–40.

    Google Scholar 

  • VAN DUIJN, P. & VAN DER PLOEG, M. (1970) Potentialities of cellulose and polyacrylamide films as vehicles in quantitative cytochemical investigations on model substances. InIntroduction to Quantitative Cytochemistry (edited by WEID, G. L. & BAHR, G. F.), pp. 232–62. New York, London: Academic Press.

    Google Scholar 

  • VANHA-PERTULA, T. & GRIMLY, P. M. (1970) Loss of proteins and other macromolecules during preparation of cell cultures for high resolution autoradiography. Quantitation by a micromethod.J. Histochem. Cytochem. 18 565–73.

    Google Scholar 

  • VAN SOEST, P. L., DE JOSSELIN DE JONG, J., LANSDORF, P. M. & VAN EWIJK, W. (1984) An automatic fluorescence micro-ELISA system for quantitative screening of hybridoma supernatants using a protein-A-B-galactosidase conjugate.Histochem. J. 16 21–35.

    Google Scholar 

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Authors and Affiliations

  1. Department of Medical Microbiology, The London School of Hygiene and Tropical Medicine, Keppel St, WC1E 7HT, London, UK

    M. R. Barer & B. S. Drasar

  2. Department of Pathology, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark

    H. Lyon

Authors
  1. M. R. Barer
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  2. H. Lyon
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  3. B. S. Drasar
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Barer, M.R., Lyon, H. & Drasar, B.S. Quantitation of dye binding by cell monolayers in a microtiter system. Histochem J 18, 122–128 (1986). https://doi.org/10.1007/BF01675366

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  • DOI: https://doi.org/10.1007/BF01675366

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