Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.

Department of Biological and Biochemical Sciences Faculty of Chemical Technology University of Pardubice Pardubice Czech Republic

Citace poskytuje Crossref.org

Cysteine conjugates of acetaminophen and p-aminophenol are potent inducers of cellular impairment in human proximal tubular kidney HK-2 cells

Structure-Guided Design of N-Methylpropargylamino-Quinazoline Derivatives as Multipotent Agents for the Treatment of Alzheimer's Disease

Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

The effect of repeated passaging on the susceptibility of human proximal tubular HK-2 cells to toxic compounds