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Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis.
Department of Molecular and Clinical Medicine Linköping University.
Department of Neuroscience and Locomotion Linköping University.
Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology .
Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Radiation Physics .
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2008 (English)In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, Vol. 70, no 6, 1059-1065 p.Article in journal (Refereed) Published
Abstract [en]

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria-neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.

Place, publisher, year, edition, pages
2008. Vol. 70, no 6, 1059-1065 p.
Keyword [en]
Cells, Cultured Electron Spin Resonance Spectroscopy/*methods Humans Neutrophils/chemistry/*metabolism Staphylococcus aureus/*physiology Staphylococcus epidermidis/*physiology Subcellular Fractions Superoxides/*analysis/chemistry/*metabolism
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-43449DOI: 10.1016/j.jbbm.2007.05.014Local ID: 73882OAI: diva2:264308
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2011-01-10

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Johansson, UnoGustafsson, HåkanÖllinger, Karin
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Faculty of Health SciencesExperimental Pathology Radiation Physics Department of Clinical Pathology and Clinical Genetics
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