Measurement of intracellular LDH activity in 96-well cultures: A rapid and automated assay for cytotoxicity studies
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The present paper describes a rapid, easy, sensitive, and automated spectrophotometric assay for intracellular lactate dehydrogenase (LDH) measurement in 96-well plates of adherent cells for cytotoxicity studies. The procedure involves “in situ” homogenization of cells, followed by measurement of LDH activity with a colorimetric method based on the reduction of a tetrazolium salt to a violet formazan by the NADH formed by LDH. Color intensity can be measured in conventional ELISA readers, and the data can be fed to an “on line” computer for rapid processing. The color absorbance measured is time- and enzyme-concentration dependent. LDH activity measured with this micromethod is coincident with that measured in larger culture plates after individual homogenization and conventional LDH measurement. The advantages of this method are the smaller number of cells required, easy automation, drastic reduction of time for processing individual wells, and the possibility of examining multiple variables in the same experiment.
Key wordsintracellular lactate dehydrogenase tetrazolium salt micromethod
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- 1.Deneke, U. Lactate dehydrogenase, colorimetric method. In: Bergmeyer, H. U.; Bergmeyer, J.; Graßl, M., eds. Methods of enzymatic analysis, 3rd ed., vol. 3. Weinheim, FRG: Verlag Chemie; 1983:133–138.Google Scholar
- 3.Gómez-Lechón, M. J.; Larrauri, A.; Donato, T., et al. Predictive value of the in vitro test for hepatotoxicity of xenobiotics. In: Guillouzo, A., ed. Liver cells and drugs, vol 164. Paris (France): Colloque INSERM/John Libbey Eurotext Ltd.; 1988:371–377.Google Scholar
- 4.Gómez-Lechón, M. J.; Castell, J. V. Efficiency of phylogenetically unrelated plasma fibronectins on hepatocyte attachment to substrate. Cienc. Biol. 8:49–56; 1983.Google Scholar
- 6.Mitchell, D. B.; Santone, K. S.; Acosta, D. Evaluation of cytotoxicity in cultured cells by enzyme leakage. J. Tissue Cult. Methods 6:113–116; 1980.Google Scholar
- 7.Wahlefeld. A. W. Lactate dehydrogenase, UV-method with L-lactate and NAD. In: Bergmeyer, H. U.; Bergmeyer, J.; Graßl, M., eds. Methods of enzymatic analysis. 3rd ed., vol. 3. Weinham, FRG: Verlag Chemie; 1983:126–133.Google Scholar