Abstract
We describe a modified enzymatic, kinetic, glutathione microassay based on the original macroassay described by Tietze and modified by Anderson. It is coupled with the Triton X-100 lysis and an acid extraction that can be performed in 96-well microtiter plates. The microassay can be read in a microplate reader equipped with a standard 405 nm filter. Intracellular glutathione levels are not significantly different when comparing the proposed Triton X-100 lysis and acid extraction method from those found with the cellular homogenization and acid extraction method typically employed. By combining a rapid sample extraction method, which can be done on the microtiter plate, with an assay based on the technology of a microplate reader, we have devised a rapid, reproducible, inexpensive and easy to use GSH microassay that can process several hundred samples daily and will be useful in studies where many replicate samples are required. We have successfully used this method to monitor glutathione status in a human cell line exposed to organochlorine pesticides and cells exposed to naturally occurring mixtures of metals extracted from aquatic sediments.
Similar content being viewed by others
References
Meister A (1985). Methods for the selective modifications of glutathione metabolism and study of glutathione transport. In: Meister A (ed), Methods in Enzymology, vol 113. New York, NY: Academic Press, pp 571-585.
Tyson C, Green CE. (1987). Cytotoxicity measures: choices and methods. In: Rauckman EJ, Padilla GM (eds), The Isolated Hepatocyte: Use in Toxicology and Xenobiotic Biotransformations. New York, NY: Academic Press, pp 119-158.
Baker MA, Cerniglia GJ, Zaman A (1990). Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 190: 360-365.
Berger SJ, Gosky D, Zborowska E, Willson, JKV, Berger N (1994). Sensitive enzymatic cycling assay for glutathione: Measurements of glutathione content and its modulation by buthionine sulfoximine in vivo and in vitro in human colon cancer. Cancer Res 54: 4077-4083.
Teitze F (1969). Enzymatic method for quantitative determination of nanogram amounts of total oxidized glutathione: Applications to mammalian blood and other tissues. Anal Biochem 27: 502-522.
Anderson ME (1985). Determination of glutathione and glutathione disulfide in biological samples. In: Meister A (ed), Methods of Enzymology, vol 113. New York, NY: Academic Press, pp 548-555.
Martin TD, Brockoff CA, Creed JT, & EMMC Methods Work Group-Method 200.7. Method 200.7. Determination of metals and trace elements in water and wastes by inductively coupled plasma-atomic emission spectrometry. Environmental Monitoring Systems Laboratory, Office of Research & Development, US Environmental Protection Agency, Cincinnati, Ohio, Revision 4.4 (May 1994). 200.7-1-200.7-57.
Rights and permissions
About this article
Cite this article
Allen, S., Shea, J.M., Felmet, T. et al. A kinetic microassay for glutathione in cells plated on 96-well microtiter plates. Methods Cell Sci 22, 305–312 (2000). https://doi.org/10.1023/A:1017585308255
Issue Date:
DOI: https://doi.org/10.1023/A:1017585308255