Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells

Čapek, Jan; Hauschke, Martina; Brůčková, Lenka; Roušar, Tomáš

doi:10.1016/j.vascn.2017.06.001

Cited by 36 publications

(26 citation statements)

References 55 publications

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“…Other fluorescent probes, such as monochlorobimane (MCB) and monobromobimane (MBB), used less frequently, also form stable fluorescent adducts with GSH, which can be determined at Ex/Em = 394/490 nm. In comparison with OPA, MCB and MBB have the substantial advantage of penetrating into the cell to react directly with cellular thiols, preventing possible GSH oxidation after cell lysis and allowing analysis by flow cytometry and fluorescence microscopy [158–161].…”

Section: Methods and Approaches To Measure Oxidative Stress In CLImentioning

confidence: 99%

Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field

Katerji

Filippova

Duerksen-Hughes

2019

Oxidative Medicine and Cellular Longevity

287

205

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Reactive oxygen species (ROS) are common by-products of normal aerobic cellular metabolism and play important physiological roles in intracellular cell signaling and homeostasis. The human body is equipped with antioxidant systems to regulate the levels of these free radicals and maintain proper physiological function. However, a condition known as oxidative stress (OS) occurs, when ROS overwhelm the body’s ability to readily detoxify them. Excessive amounts of free radicals generated under OS conditions cause oxidative damage to proteins, lipids, and nucleic acids, severely compromising cell health and contributing to disease development, including cancer. Biomarkers of OS can therefore be exploited as important tools in the assessment of disease status in humans. In the present review, we discuss different approaches used for the evaluation of OS in clinical samples. The described methods are limited in their ability to reflect on OS only partially, revealing the need of more integrative approaches examining both pro- and antioxidant reactions with higher sensitivity to physiological/pathological alternations. We also provide an overview of recent findings of OS in patients with different types of cancer. Identification of OS biomarkers in clinical samples of cancer patients and defining their roles in carcinogenesis hold great promise in promoting the development of targeted therapeutic approaches and diagnostic strategies assessing disease status. However, considerable data variability across laboratories makes it difficult to draw general conclusions on the significance of these OS biomarkers. To our knowledge, no adequate comparison has yet been performed between different biomarkers and the methodologies used to measure them, making it difficult to conduct a meta-analysis of findings from different groups. A critical evaluation and adaptation of proposed methodologies available in the literature should therefore be undertaken, to enable the investigators to choose the most suitable procedure for each chosen biomarker.

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Section: Methods and Approaches To Measure Oxidative Stress In CLImentioning

confidence: 99%

Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field

Katerji

Filippova

Duerksen-Hughes

2019

Oxidative Medicine and Cellular Longevity

287

205

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“…Glutathione levels were measured using the monochlorobimane spectrofluorometric assay 70 . After cell treatment, 20 µL of the bimane solution was added to cells to obtain the final concentration 40 µM and spectrofluorometric measurement was started immediately.…”

Section: Methodsmentioning

confidence: 99%

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

Majtnerová

Čapek

Petira

et al. 2021

Sci Rep

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At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6–48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

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“…Glutathione levels were measured using the monochlorobimane spectro uorometric assay 67 . After cell treatment, 20 µL of the bimane solution was added to cells to obtain the nal concentration 40 µM and spectro uorometric measurement was started immediately.…”

Section: Glutathione Assaymentioning

confidence: 99%

Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

Majtnerová

Čapek

Petira

et al. 2021

Preprint

Self Cite

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At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods has used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in intact cells. We used HepG2 and HK‑2 cells cultured in 96-well plates which were treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Then, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting apoptosis (i.e. TUNEL, DNA ladder, caspase activity). We found that the developed assay provides results of same sensitivity as TUNEL assay but the advantages of the spectrofluorometric assay are fast processing, low-cost and high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

show abstract

Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells

Cited by 36 publications

References 55 publications

Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field

Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

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