Comparison of different methods for measuring the superoxide radical by EPR spectroscopy in buffer, cell lysates and cells

Autor:	Bernard Gallez, Pierre Sonveaux, Benoit Driesschaert, Samantha Scheinok, Philippe Levêque
Přispěvatelé:	UCL - SSS/LDRI - Louvain Drug Research Institute, UCL - SSS/IREC/FATH - Pôle de Pharmacologie et thérapeutique
Rok vydání:	2018
Předmět:	0301 basic medicine Cell Extracts nitrones spin-trapping Buffers Photochemistry Biochemistry Article law.invention Nitrone 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Hydroxylamine nitroxides law Superoxides Animals Reactivity (chemistry) Electron paramagnetic resonance ESR chemistry.chemical_classification Spin trapping Molecular Structure Superoxide trityls Electron Spin Resonance Spectroscopy hydroxylamines General Medicine 030104 developmental biology RAW 264.7 Cells chemistry 030220 oncology & carcinogenesis Molecular Probes Phorbol Hydroxyl radical EPR superoxide
Zdroj:	Free radical research, p. 1-15 (2018)
ISSN:	1029-2470
Popis:	As superoxide anion is of keen interest in biomedical research, it is highly desirable to have a technique allowing its detection sensitively and specifically in biological media. If electron paramagnetic resonance (EPR) techniques and probes have been individually described in the literature, there is actually no comparison of these techniques in the same conditions that may help guiding researchers for selecting the most appropriate approach. The aim of the present study was to compare different EPR strategies in terms of sensitivity and specificity to detect superoxide (versus hydroxyl radical). Three main classes of EPR probes were used, including paramagnetic superoxide scavengers (such as nitroxides TEMPOL and mitoTEMPO as well as trityl CT-03), a spin trap (DIPPMPO), and diamagnetic superoxide scavengers (such as cyclic hydroxylamines CMH and mitoTEMPO-H). We analyzed the reactivity of the different probes in the presence of a constant production of superoxide or hydroxyl radical in buffers and in cell lysates. We also assessed the performances of the different probes to detect superoxide produced by RAW264.7 macrophages stimulated by phorbol 12-myristate 13-acetate (PMA). In our conditions and models, we found that nitroxides were not specific for superoxide. CT-03 was specific, but the sensitivity of detection was low. Comparatively, we found that nitrone DIPPMPO and cyclic hydroxylamine CMH were good candidates to sensitively and specifically detect superoxide in complex biological media, CMH offering the best sensitivity.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::25e8f564720a6c65e352d47636ae554c https://pubmed.ncbi.nlm.nih.gov/30362382 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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