Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species in Staphylococcus aureus
电子顺磁共振（EPR）光谱法检测金黄色葡萄球菌中的活性氧类物质
发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1586 浏览次数: 11274
评审: Fanglian HeBenoit Chassaing
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
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Abstract
Under aerobic conditions, Staphylococcus aureus (S. aureus) primarily metabolizes glucose to acetic acid. Although normally S. aureus is able to re-utilize acetate as a carbon source following glucose exhaustion, significantly high levels of acetate in the culture media may not only be growth inhibitory but also potentiates cell death in stationary phase cultures by a mechanism dependent on cytoplasmic acidification. One consequence of acetic acid toxicity is the production of reactive oxygen species (ROS). The present protocol describes the detection of ROS in S. aureus undergoing cell death by electron paramagnetic resonance (EPR) spectroscopy. Using 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) as a cell permeable spin probe, we demonstrate the detection of various oxygen radicals generated by bacteria. Although standardized for S. aureus, the methods described here should be easily adapted for other bacterial species. This protocol is adapted from Thomas et al. (2014) and Thomas et al. (2010).
Keywords: EPR (EPR)
Staphylococcus aureus (金黄色葡萄球菌)
Reactive oxygen species (活性氧)
Spectroscopy (光谱)
Materials and Reagents
- Staphylococcus aureus
- Bacto tryptic soy broth without dextrose (TSB) (BD Diagnostic Systems, catalog number: DF0862178 )
- Glucose (Sigma-Aldrich, catalog number: G8270 ).
- 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Noxygen Science Transfer & Diagnostics GmbH, catalog number: NOX-2.2-100mg )
- Superoxide dismutase (SOD) (Sigma-Aldrich, catalog number: S7571 )
- Dimethyl thiourea (DMTU) (Sigma-Aldrich, catalog number: D188700 )
- Critoseal (Thermo Fisher Scientific, catalog number: 0267620 )
- 5 µM DETC (Noxygen, Catalog number: NOX-10.1 )
- 25 µM deferoxamine (Noxygen, Catalog number: NOX-10.1)
- Culture flask (250 ml)
- Culture tubes
- 1.5 ml Eppendorf tubes
- Krebs-HEPES buffer (KDD buffer) (see Recipes)
Equipment
- 37 °C shaker-incubator (250 rpm per min)
- Leica Biosystems Critoseal capillary tube sealant (Leica Microsystems, catalog number: MS215003A )
- Bruker e-Scan EPR Spectrometer and Noxygen Temperature Controller Bio-I (Bruker, model: NOX-E.11-ESR )
- Micropipettes (50 µl, EPR tubes) (Noxygen Science Transfer & Diagnostics GmbH, catalog number: MS215003A )
- Spectrophotometer
- Vortex-Genie 2
Software
- Bruker WinEPR Data Processing software
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Thomas, V. C., Chaudhari, S. S., Jones, J., Zimmerman, M. C. and Bayles, K. W. (2015). Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species in Staphylococcus aureus. Bio-protocol 5(17): e1586. DOI: 10.21769/BioProtoc.1586.
- Thomas, V. C., Sadykov, M. R., Chaudhari, S. S., Jones, J., Endres, J. L., Widhelm, T. J., Ahn, J. S., Jawa, R. S., Zimmerman, M. C. and Bayles, K. W. (2014). A central role for carbon-overflow pathways in the modulation of bacterial cell death. PLoS Pathog 10(6): e1004205.
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分类
微生物学 > 微生物生物化学 > 其它化合物
生物化学 > 其它化合物 > 活性氧
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