title: Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps.
abstract: Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.
authors.affiliation: LCBPT, UMR 8601 CNRS - Paris Descartes University , Sorbonne Paris Cité, Paris , France.
authors.forename: K
authors.initials: K
authors.lastname: Abbas
authors.forename: M
authors.initials: M
authors.lastname: Hardy
authors.forename: F
authors.initials: F
authors.lastname: Poulhès
authors.forename: H
authors.initials: H
authors.lastname: Karoui
authors.forename: P
authors.initials: P
authors.lastname: Tordo
authors.forename: O
authors.initials: O
authors.lastname: Ouari
authors.forename: F
authors.initials: F
authors.lastname: Peyrot
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000737:chemistry; D002448:Cell Adhesion; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals; D051379:Mice; D009589:Nitrogen Oxides / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry; D000067996:RAW 264.7 Cells; D017382:Reactive Oxygen Species / Q000378:metabolism; D013113:Spin Labels; D019097:Spin Trapping / Q000379:methods*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D047392:beta-Cyclodextrins / Q000737:chemistry
chemical_list: C510912:(diethoxyphosphoryl)-5-methylpyrroline N-oxide; C512236:5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide; D000975:Antioxidants; C000607737:CD-DIPPMPO; D003497:Cyclic N-Oxides; D005609:Free Radicals; C000607736:Mito-DIPPMPO; D009589:Nitrogen Oxides; D063065:Organophosphonates; D011758:Pyrroles; D017382:Reactive Oxygen Species; D013113:Spin Labels; D047392:beta-Cyclodextrins; C015388:nitrones; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen
keywords: CD-DIPPMPO; EPR spectroscopy; RAW macrophages; free radical; spin trapping