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Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.
The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 °C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 °C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.
While measures of oxidative stress and reactive oxygen species are important to the study of diverse diseases across all organ systems, the detection of reactive oxygen species (ROS) is challenging due to a short half-life and high reactivity. An electron paramagnetic resonance (EPR) technique is the most unambiguous method for detecting free radicals. Spin probes have advantages over the more commonly used fluorescent probes. Though fluorescent probes are relatively inexpensive and easy to use and provide rapid, sensitive detection of ROS, they do have serious limitations due to artifactual signals, an inability to calculate ROS concentrations, and a general lack of ....
All animal studies were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
1. Preparation of Reagents
Superoxide detection using CMH was validated using the X/XO superoxide generating system to demonstrate that the nitroxide (CM.) signal was fully inhibited by SOD, while catalase had no effect (Figure 1A). The total, extracellular superoxide was then evaluated in RAW 264.7 cells by incubating cells with the cell-permeable CMH spin probe +/- SOD pretreatment. The nitroxide concentration was measured in both the cell suspension and buffer, which.......
The assessment of free radical production in biological settings is important in understanding redox regulated signaling in health and disease, but the measure of these species is highly challenging due to the short half-life of free radical species and technical limitations with commonly used methods. EPR is a valuable and powerful tool in redox biology, as it is the only unambiguous method for detecting free radicals. In this project, we demonstrate practical EPR methods for designing experiments and preparing samples .......
This work was supported by the University of Colorado School of Medicine Dean's Strategic Research Infrastructure award, R01 HL086680-09 and 1R35HL139726-01, to E.N.G. and UCD CFReT fellowship award (HE). The authors thank Dr. Sandra Eaton and Dr. Gareth Eaton (University of Denver), Dr. Gerald Rosen and Dr. Joseph P. Kao (University of Maryland), and Dr. Sujatha Venkataraman (University of Colorado Denver) for helpful discussions, and Joanne Maltzahn, Ashley Trumpie and Ivy McDermott (University of Colorado Denver) for technical support.
....Name | Company | Catalog Number | Comments |
DMEM | LifeTech | 10566-016 | cell culture media |
Diethylenetriaminepentaacetic acid (DTPA) | Sigma Aldrich | D6518-5G | |
sodium chloride (NaCl) | Fisher Scientific | BP358-212 | used to prepare 50 mM phosphate saline buffer according to Sigma aldrish |
potassium phosphate dibasic (HK2PO4 ) | Fisher Scientific | BP363-500 | used to prepare 50 mM phosphate saline buffer according to Sigma aldrish |
potassium phosphate monobasic (KH2PO4 ) | Sigma Aldrich | P-5379 | used to prepare 50 mM phosphate saline buffer according to Sigma aldrish |
Krebs-Henseleit buffer (KHB) | (Alfa Aesar, Hill) | J67820 | |
Bovine erythrocyte superoxide dismutase (SOD) | Sigma Aldrich | S7571-30KU | |
Phorbol 12-myristate 13-acetate (PMA) | Sigma Aldrich | P1585-1MG | Dissolve in DMSO |
Antimycin A (AA) | Sigma Aldrich | A8674-25MG | Dissolve in Ethanol and store in glass vials(MW used is the averaged molecular weights for four lots) |
1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine . HCl (CMH) | Enzo Life Sciences | ALX-430-117-M050 | |
1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine . HCl (CPH) | Enzo Life Sciences | ALX-430-078-M250 | |
1-Hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine, 1-Hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido]piperidinium dichloride ( mito-TEMPO-H) | Enzo Life Sciences | ALX-430-171-M005 | |
1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride . HCl (CAT1H) | Enzo Life Sciences | ALX-430-131-M250 | |
Heparin | Sagent Pharmaceuticals | NDC 25021-400-10 | |
Diphenyliodonium chloride | Sigma Aldrich | 43088 | |
Deferoxamin mesylate salt | Sigma Aldrich | D9533-1G | |
Critoseal | Leica | 39215003 | |
BRAND disposable BLAUBRAND micropipettes, intraMark | Sigma Aldrich | 708733 | Capillaries |
PTFE FRACTIONAL FLUOROPOLYMER TUBING 3/16” OD x 1/8” ID | NORELL | 1598774A | Teflon tubing |
SILICONE RUBBER STOPPERS FOR NMR SAMPLE TUBES FOR THIN WALL TUBES HAVING AN OD OF 4mm-5mm (3.2mm TO 4.2mm ID) TS-4-5-SR | NORELL | 94987 | |
EMXnano Bench-Top EPR spectrometer | Bruker BioSpin GmbH | E7004002 | |
EMX NANO TISSUE CELL | Bruker BioSpin GmbH | E7004542 |
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