Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
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 assa...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-02-01
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| Series: | Antioxidants |
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| Online Access: | https://www.mdpi.com/2076-3921/11/2/391 |
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| author | Rezeda A. Ishkaeva Mohamed Zoughaib Alexander V. Laikov Plamena R. Angelova Timur I. Abdullin |
| author_facet | Rezeda A. Ishkaeva Mohamed Zoughaib Alexander V. Laikov Plamena R. Angelova Timur I. Abdullin |
| author_sort | Rezeda A. Ishkaeva |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T22:43:18Z |
| format | Article |
| id | doaj.art-266e3c13740c4a9dad42cbc723a92236 |
| institution | Directory Open Access Journal |
| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-09T22:43:18Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-266e3c13740c4a9dad42cbc723a922362023-11-23T18:33:06ZengMDPI AGAntioxidants2076-39212022-02-0111239110.3390/antiox11020391Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate AssayRezeda A. Ishkaeva0Mohamed Zoughaib1Alexander V. Laikov2Plamena R. Angelova3Timur I. Abdullin4Department of Biochemistry, Biotechnology and Pharmacology, Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., 420008 Kazan, RussiaDepartment of Biochemistry, Biotechnology and Pharmacology, Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., 420008 Kazan, RussiaDepartment of Biochemistry, Biotechnology and Pharmacology, Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., 420008 Kazan, RussiaDepartment of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UKDepartment of Biochemistry, Biotechnology and Pharmacology, Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., 420008 Kazan, RussiaThiol 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.https://www.mdpi.com/2076-3921/11/2/391glutathione statusoxidative statusmammalian cellsmonochlorobimanemicroplate assayredox-modulating factors |
| spellingShingle | Rezeda A. Ishkaeva Mohamed Zoughaib Alexander V. Laikov Plamena R. Angelova Timur I. Abdullin Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay Antioxidants glutathione status oxidative status mammalian cells monochlorobimane microplate assay redox-modulating factors |
| title | Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay |
| title_full | Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay |
| title_fullStr | Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay |
| title_full_unstemmed | Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay |
| title_short | Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay |
| title_sort | probing cell redox state and glutathione modulating factors using a monochlorobimane based microplate assay |
| topic | glutathione status oxidative status mammalian cells monochlorobimane microplate assay redox-modulating factors |
| url | https://www.mdpi.com/2076-3921/11/2/391 |
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