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.

• Klíčová slova
• Cell impairment, Fluorescence, Glutathione assay, Monochlorobimane, Ortho-phthalaldehyde,
• MeSH
• biotest ekonomika   metody   MeSH
• buněčné linie MeSH
• cisplatina toxicita   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie ekonomika   metody   MeSH
• glutathion analýza   metabolismus   MeSH
• lidé MeSH
• malonáty toxicita   MeSH
• o-ftalaldehyd chemie   MeSH
• průtoková cytometrie MeSH
• pyrazoly chemie   MeSH
• senzitivita a specificita MeSH
• studie proveditelnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cisplatina MeSH
• diethyl malonate MeSH Prohlížeč
• fluorescenční barviva MeSH
• glutathion MeSH
• malonáty MeSH
• monochlorobimane MeSH Prohlížeč
• o-ftalaldehyd MeSH
• pyrazoly MeSH

Redox regulations and antioxidant defence play a central role in the acclimation of plants to their environment. Glutathione represents an essential component of the cellular antioxidant defence system, which keeps levels of reactive oxygen species (ROS) under control. High-performance liquid chromatography (HPLC) separation with fluorescence detection is a sensitive method that enables analysis of reduced and oxidised glutathione levels in small samples of plant tissues or plant cell culture. We aimed to optimise the method to obtain more accurate information about the total level of glutathione and the proportion of the reduced form (GSH) by choosing the most suitable reduction reagent and the conditions under which the reduction occurs. The applicability of the developed method was verified by analysing tobacco cells treated with hydrogen peroxide, which caused a decrease in the GSH/total glutathione ratio. Significant changes in the level of glutathione as well as in the GSH/total glutathione ratio were also observed during tobacco cell culture development.

• Klíčová slova
• Glutathione, HPLC analysis, Monochlorobimane reagent, Nicotiana tabacum, tris(2-carboxyethyl)phosphine,
• MeSH
• glutathion * analýza   MeSH
• kalibrace MeSH
• kultivované buňky MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• peroxid vodíku farmakologie   MeSH
• rostlinné buňky * MeSH
• tabák * genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glutathion * MeSH
• peroxid vodíku MeSH