Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K
Summary
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.
Abstract
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.
Introduction
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 specificity1.
To facilitate the use of EPR for biological studies, a variety of spin probes have been synthesized that can measure a range of biologically relevant free radical species as well as pO2, pH, and redox states2,3,4,5,6,7. Spin traps have also been developed to capture short-lived radicals and form long-living adducts, which facilitates detection by EPR8. Both classes (spin probes and spin traps) have advantages and limitations. One commonly used class of spin probes are cyclic hydroxylamines, which are EPR-silent and react with short-lived radicals to form a stable nitroxide. Cyclic hydroxylamines react with superoxide 100 times faster than spin traps, enabling them to compete with cellular antioxidants, but they lack specificity and require the use of appropriate controls and inhibitors to identify the radical species or source responsible for the nitroxide signal. While spin traps exhibit specificity, with distinct spectral patterns depending on the trapped species, they have slow kinetics for superoxide spin trapping and are prone to biodegradation of the radical adducts. Applications for spin trapping have been well-documented in biomedical research9,10,11,12,13.
The goal of this project is to demonstrate practical EPR methods for designing experiments and preparing samples to detect superoxide using spin probes in different cellular compartments in vitro and in different tissue compartments in vivo. Several manuscripts have published protocols relevant to these goals, using cell-permeable, cell-impermeable, and mitochondrial targeted spin probes to target different cellular compartments in vitro and process tissue for analysis in mouse models14,15. We build upon this body of literature by validating an approach to measure superoxide using a 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) spin probe in different cellular compartments in vitro to ensure accurate measurements, highlighting potential technical problems that may skew results. We also provide methods to perform EPR measurements in blood, bronchoalveolar lavage fluid, and lung tissue using the CMH spin probe. These studies compare different methods to process the tissues as well as present a method to inject another spin probe, CPH, into mice prior to harvesting tissue. Finally, we develop a practical method to store samples in polytetrafluoroethylene (PTFE) tubing to allow for the storage and transfer of samples before EPR measurements at 77 K.
Protocol
Representative Results
Discussion
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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