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Kinetic analysis of glutathione in anchored cells with monochlorobimane

Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station 77843-4458, USA.
Cytometry 03/1995; 19(3):226-34. DOI: 10.1002/cyto.990190306
Source: PubMed

ABSTRACT A method for the measurement of intracellular glutathione content and glutathione S-transferase activity with monochlorobimane in adherent cells is described. The method involves the kinetic analysis of monochlorobimane conjugation to glutathione over a relatively short period of time. This permits extrapolation over time for determination of equilibrium fluorescence intensity (relative glutathione level) from scan intensity data that follows first-order kinetics, minimizing problems commonly associated with the use of monochlorobimane. By using measured fluorescence intensity values from glutathione standards, a suspension calibration curve was generated and, subsequently, was used to determine the photomultiplier tube saturation rate. A theoretical intracellular calibration curve was then generated to quantify glutathione content in cells. This method was also applied to study the changes in glutathione in a variety of rodent and human cell lines and in selected cocultures of cells exhibiting similar or different glutathione levels. Comparison of the glutathione levels obtained with monochlorobimane and a standard colorimetric method (GSH-400) indicated good correlation between the two methods. These studies support the use of laser cytometry for measuring intracellular glutathione with monochlorobimane as well as changes in glutathione occurring in cells that establish physical contacts with other cells. Laser cytometric analysis of glutathione in anchored cells also provides opportunities to monitor individual cellular responses to a variety of experimental manipulations, such as responses to various toxic insults or the protective effects of gap junction-mediated intercellular communication.

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    • "ocytes were cultured in DMEM for 18 – 24 h before study . Intracellular [ GSH ] was measured by fluorescence microscopy using the probe monochlorobimane ( mBCl ; Molecular Probes , Carlsbad , CA , USA ) . The cell - permeant mBCl is non - fluorescent but forms a fluorescent adduct with GSH in a reaction catalysed by glutathione - S - transferase ( Barhoumi et al . 1995 ; Li et al . 2003 , 2007 , 2008a ) . Glass coverslips with attached myocytes were transferred to a culture dish filled with loading solution containing ( in mmol l"
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    • "The specificity of monochlorobimane for GSH results from the fact that it is conjugated to GSH by glutathione S-transferase and has low nonenzymatic reactivity toward GSH and other thiols [66] [67]. Comparisons of cellular GSH levels determined by the monochlorobimane assay and by other assays show excellent correlation [6] [70]. The fact that we observed near complete signal depletion when cells were incubated with BSO (buthionine sulfoximine), a specific and irreversible inhibitor of g-glutamyl-cysteine synthetase (an enzyme crucial for GSH biosynthesis), confirms the specificity of this assay in our system. "
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