Abstract
Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.
Highlights
Thiol-containing biomolecules are a key component of protecting antioxidant and regulatory systems in mammalian cells
3T3 mouse embryonic fibroblasts were used as model mammalian cells sensitive to redox-modulating factors [28,29,30]
A reliable in situ assessment of GSH modulation in living cells is of considerable interest in research related to redox homeostasis, GSH-replenishing and cytoprotective therapy
Summary
Thiol-containing biomolecules are a key component of protecting antioxidant and regulatory systems in mammalian cells. The tripeptide glutathione (L-γ-glutamyl-L-cysteinylglycine) is the predominant thiol with an intracellular concentration greatly exceeding that of sulfur amino acids, including the immediate precursor L-cysteine [1,2]. It has multiple activities that rely on the reactions of cysteine thiol catalyzed by a series of glutathionedependent enzymes. These reactions involve the elimination of reactive oxygen and nitrogen species (ROS and RNS), inactivation of harmful electrophilic compounds and α-oxoaldehydes, as well as redox signaling in cells [3]. Regulatory mechanisms of the GSH/GSSG couple are based on the direct suppression of ROS/RNS-mediated signaling and modulation of the activity of redox-sensitive proteins via thiol–disulfide conversions of cysteine residues including the formation of mixed cysteine-glutathione disulfide. The depletion of GSH due to an abnormal production of ROS/RNS and impaired antiradical defense underlies a series of pathological conditions such as excessive apoptosis, degeneration and chronic inflammation [6], as well as tumor transformation [7] and viral propagation [8]
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