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 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.
All animal studies were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
1. Preparation of Reagents
2. Detection of Superoxide in vitro
3. EPR Measurements in Fluids
4. EPR Measurements on Lung Tissue
5. Data Analysis
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 demonstrated that the values in the two sample types were similar due to the permeable nature and rapid equilibration of the spin probe (Figure 1B). The nitroxide radical signal increased in RAW 264.7 cells stimulated with PMA compared to control cells. This signal was significantly attenuated in cells pretreated with cell-impermeable SOD (Figure 1C). Each color represents wells tested on different days, demonstrating the consistency of data collected on specific days and reproducibility of the results across time. The concentration of extracellular superoxide was determined by subtracting the signal in PMA cells pretreated with SOD from the signal after PMA in the absence of SOD (T). The remaining signal was attributed to intracellular superoxide (Figure 1C). Figure 1D illustrates the calculation of total and extracellular superoxide. (E) The intracellular signal was confirmed in PMA-treated cells after removal of the media and by the effect of PEG-SOD on the signal. In this graph, in contrast to (C), the CMH blank was not subtracted from the measurements, and the raw data is shown.
Mitochondrial superoxide in RAW 264.7 cells was detected using the EPR spin probe mito-TEMPO-H, which accumulates in mitochondia. (A) Representative EPR spectra for the baseline mito-TEMPO-H signal in buffer, the increased mito-TEMPO-H signal in control cells (Con), and the further enhanced signal in cells stimulated with the mitochondrial inhibitor Antimycin A (AA). The increase in the signal was attributed to the mitochondrial superoxide based on our previous study showing that SOD2 overexpression significantly attenuated measurements with mito-TEMPO-H10. In Figure 2B, the mitochondrial nitroxide concentration was determined by subtracting the mito-TEMPO-H signal in time-matched buffer from the cell measurements. The CM. signal obtained at low temperatures in RAW 264.7 cells after stimulation with PMA in the presence and absence of SOD. (Figure 3A) The CM. signal was attenuated in the presence of SOD, consistent with the room temperature data (Figure 1). Figure 3B shows the photograph of PTFE tubing with the stoppers used to collect data at 77 K for cells and in vivo samples. Superoxide production was detected in blood and BALF using the CMH spin probe. Blood or BALF samples were collected from PBS- and Bleo-treated mice and incubated immediately with CMH. The samples were transferred to the PTFE tubing and flash frozen, and EPR data was collected at 77 K. The concentration of nitroxide (CM.) accumulated in blood incubated with CMH (0.2 mM) at 37 degrees for 10 min (Figure 4A). Nitroxide (CM.) concentration from BALF incubated for 50 min (Figure 4B). Nitroxide concentration represents the concentration of (CM.) accumulated in volume of blood or BALF used in the experiment.
Three methods have been tested to evaluate several published techniques for tissue preservation and administration of spin probes ex vivo vs. in vivo. To perform EPR measurements on lung tissue, we first used flash frozen lung tissue from control or injured mice. Figure 5A shows the total CM. signal in the supernatant of a small piece of lung tissue incubated at 37 °C with CMH in PBS- and Bleo-treated mice, respectively. Due to heterogeneity of the lung injury after Bleo treatment, it is recommended to cut pieces from different regions of the lung and average several measurements to provide a more representative value. Alternatively, one can homogenize the entire lung and use one sample of this homogenate. Data collected at 77 K using PTFE tubing and finger dewar. Figure 5B shows representative spectra of nitroxide (CM.) signals from PBS- and Bleo-treated mice, respectively.
One limitation to treating lung tissue ex vivo is that it is not possible to reliably distinguish extracellular from intracellular superoxide due to the processing of the tissue that disrupts cell membranes. If this information is important to the experimental question, it can be addressed by using the in vivo CPH instillation method described below. Frozen tissue cannot be used to assess mitochondrial superoxide; though, for this measurement, the protocol can be adapted to use mito-TEMPO-H in the tissue or freshly isolated mitochondria.
As a second method for EPR measurements in lung tissue, fresh tissue was homogenized in sucrose buffer. The lung homogenate was incubated with CMH probe in KHB buffer containing DTPA. EPR measurements were carried out at RT. Figure 6A demonstrates the increase in CM. with Bleo. We presented an additional testing using different inhibitors that can be used to determine the species that contribute to the CM. signal. To elucidate the origin of CM. signal generated from lung tissue, we pretreated the lung homogenates with several scavengers and enzymes inhibitors. Lung homogenates were incubated with CMH in the absence or the presence of SOD, deferoxamine (DFO), and diphenyliodonium chloride (DIP) to account (respectively) for the contributions from superoxide, iron, or superoxide generated from flavin-containing enzymes (Figure 6B). This approach can be adapted to assess the specific radical species generated in a system or elucidate the contribution of other enzymatic sources (e.g., NOX, eNOS, or xanthine oxidase).
Mice were injected with CPH spin probes (20 mg/kg) via the retroorbital route to perform EPR measurements in vivo. It is unknown whether CMH can be safely administered to animals, while the CPH probe has been reported to be non-toxic; thus, we selected CPH for the in vivo experiments. Lung tissues were harvested and flash frozen in liquid nitrogen 1 h after circulation of CPH probes. Mice can be simultaneously treated with specific antioxidants to differentiate the species responsible for the signal. Figure 7A shows the higher CP. signal in Bleo-treated mice compared to control mice. Representative spectra of lung tissue from control and Bleo-treated mice are shown in Figure 7B. A mixed EPR spectra of CP. and ascorbic acid radical was observed. The values reported in Figure 7A are the concentrations of CP. components. Data were collected at RT using the tissue cell.
Figure 1: Detection of superoxide in different cell compartments. (A) EPR spectra generated by 0.25 mM CMH in 0.5 mM hypoxanthine/xanthine oxidase (8 mU/mL) with and without SOD (30 U/mL). (B) RAW 264.7 cells (1 x 106 cells/well) were stimulated with 10 µM PMA in the presence of CMH for 50 min at 37 °C and nitroxide concentration (µM) detected in cell suspension (cells + buffer) and buffer collected from treated cells. (C) RAW 264.7 cells were stimulated with PMA vs. vehicle control (Con). One set of cells were pretreated for 10 min with 30 U/mL cell-impermeable SOD (PMA + SOD). Each color represents data from different experimental days and each point represents cells from an individual well. The nitroxide signal in a time-matched blank with CMH in KHB was subtracted from each signal to obtain final values. (D) Calculation of total and extracellular superoxide in PMA stimulated cells; T = total superoxide, EC = extracellular superoxide (SOD inhibitable signal). (E) To evaluate the intracellular superoxide signal (IC), the signal in buffer after PMA + SOD was compared to PMA-treated cells after the removal of buffer. To confirm, wells were pretreated with 60 U/mL cell-permeable PEG-SOD for 1.5 hours to determine the intracellular SOD inhibitable. The time-matched CMH blank is shown, and data reflect absolute nitroxide signal. Data expressed as mean ± SEM. Please click here to view a larger version of this figure.
Figure 2: Detection of mitochondrial superoxide in RAW cells stimulated with antimycin A. (A) Representative spectra of the mitochondrial-specific EPR spin probe, 0.25 mM mito-TEMPO-H in RAW 264.7 cells without (Con) or with 25 µM antimycin A (AA) for 50 min at 37 °C. (B) CM. concentration (µM) in cells treated with AA compared to control. The nitroxide signal in a time-matched mito-TEMPO-H blank was subtracted from total signal to obtain final values. Data expressed as mean ± SEM. Please click here to view a larger version of this figure.
Figure 3: Detection of superoxide in RAW 264.7 cells at 77K. (A) RAW 264.7 cells stimulated with 10 µM PMA and EPR spin probe, CMH 0.25 mM (50 min at 37 °C) with (black) or without (red) pretreatment with 30 U/mL SOD. 100 µL of supernatant was loaded in a 1-inch in length piece of PTFE tubing, then flash frozen in liquid nitrogen. The stoppers were removed, and frozen PTFE tubing was placed in the finger dewar for data acquisition at 77 K. (B) A photo of PTFE tubing and stoppers. Please click here to view a larger version of this figure.
Figure 4: EPR measurements in blood and BALF from control and bleomycin-treated mice. Mice were treated with a single dose of intratracheal bleomycin (IT Bleo) (100 µL at 1 U/mL) or PBS vehicle. At 7 days, mice were anesthetized and euthanized. Blood was collected via right ventricular puncture into a syringe coated with 1000 USP/mL heparin containing 100 µM DTPA. Bronchoalveolar lavage fluid (BALF) was collected by lavaging the lungs with 1 mL of 100 µM DTPA in PBS. Blood and BALF were incubated for 10 or 50 min, respectively, with 0.2 mM CMH at 37 °C. 150 µL of blood or BALF was loaded in PTFE tubing flash frozen in liquid nitrogen and EPR data collected at 77 K using a finger dewar. Data show nitroxide concentrations in (A) blood and (B) BALF from PBS- and Bleo-treated mice (n = 4-6). Data expressed as mean ± SEM. (C) Representative spectra of nitroxide in blood from PBS- and Bleo-treated mice. Please click here to view a larger version of this figure.
Figure 5: EPR measurements in flash frozen lung tissue. Mice were treated with a single dose of intratracheal bleomycin (IT bleo) (100 µL at 1 U/mL) or PBS vehicle. At 7 days, the lungs were flushed with cold PBS to remove blood and flash frozen in liquid nitrogen. 5-15 mg of flash-frozen lung tissue was incubated with 0.2 mM CMH in KHB containing 100 µM in 200 µL of total volume for 1 h at 37° C. Supernatant was collected and placed in PTFE tubing and run at 77 K in the finger dewar. (A) Nitroxide concentration (µM of nitroxide normalized to 1 mg of tissue). Data represent the average of 2-3 measurements for each lung. Data expressed as mean ± SEM. (B) Representative spectra of nitroxide in lung tissue from PBS- and Bleo-treated mice. Please click here to view a larger version of this figure.
Figure 6: EPR measurements in lung tissue preserved in sucrose buffer. Mice were treated with a single dose of intratracheal bleomycin (100 µL at 1 U/mL). At 7 days post-treatment, the lungs were flushed with cold PBS to remove blood, and fresh lung tissue was homogenized in Tris-EDTA buffer containing 0.25 mM sucrose at a 1:6 lung weight/buffer volume (mg/µL) ratio. 50 µL of lung homogenate was preincubated with KHB with or without the following inhibitors for 20 min at 37 °C: SOD (100 U/mL), deferoxamine (DFO; 800 µM), and diphenyliodonium chloride (DIP; 100 μM) followed by incubation with 0.2 mM CMH in KHB containing 100 µM DTPA for 20 min at 37 °C. Data was obtained at RT using EPR capillary tubes. (A) Nitroxide concentration in lungs from PBS- and Bleo-treated mice. (B) Nitroxide concentration in Bleo lungs in the absence or the presence of inhibitors (n=3). Data expressed as mean ± SEM. Please click here to view a larger version of this figure.
Figure 7: EPR measurements in lung tissue from mice injected with CPH spin probe. 100 µL of CPH was administered via retroorbital injection for a final concentration of 20 mg of CPH per kg of body weight. After 1 h of circulation, mice were euthanized, lungs were flushed with 10 mL of cold PBS via the right ventricle, and lung tissue was flash frozen. 20 to 30 mg of lung tissue was placed in tissue cell and EPR measurements performed at RT. (A) Data expressed as spins/mg. (B) Representative spectra of nitroxide signal in PBS and Bleo lung tissues (* indicates the overlap with ascorbic acid radical). Data expressed as mean ± SEM. Please click here to view a larger version of this figure.
Inhibitors | Species |
Superoxide dismutase (SOD) | Extracellular superoxide |
Superoxide dismutase–polyethylene glycol (PEG-SOD) | Intracellular superoxide |
Catalase | Hydrogen peroxide based radicals |
Urate | Peroxynitrate |
Ethanol and DMSO | Hydroxyl radical |
Metal chelators | Metal ions (iron and copper) |
Table 1. Common inhibitors used to distinguish species responsible for spin probe oxidation.
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 to detect ROS using spin probes in different cellular compartments in vitro and different tissue compartments in vivo. We also provide practical methods to handle biological samples and store samples to improve efficiency.
Spin probes react efficiently with ROS and produce a stable nitroxide radical that can be detected with EPR. Several derivatives of the spin probe (cyclic hydroxylamine) have been synthesized with different permeability characteristics, which make them suitable for detecting free radical production in different cellular compartments10. This protocol utilized the cell-permeable spin probe, CMH; though, the impermeable spin probe 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride HCl (CAT1H) can be used to detect extracellular superoxide. Similar to our prior study in human lymphoblast cell lines18, we were able to validate use of the permeable CMH spin probe with impermeable SOD and cell permeable PEG-SOD in RAW264.7 cells (a mouse lung macrophage cell line) stimulated with PMA to differentiate between extracellular and intracellular superoxide.
We also validated the rapid equilibration of CMH between the intra- and extra-cellular compartments, and we also found that the superoxide signal in cells drops significantly after washing the cells only once with KHB (data not shown). We confirmed utility of the mitochondrial specific spin probe mito-TEMPO-H in RAW 264.7 cells to measure the increased mitochondrial superoxide generated upon stimulation with mitochondrial electron transport chain inhibitor antimycin A. The specific contribution of mitochondrial superoxide production to the mito-TEMPO-H has been previously demonstrated and can be validated in experiments using isolated fresh mitochondria or systems with mitochondrial superoxide dismutase MnSOD (SOD2) overexpression10.
The assessment of ROS production in vivo is particularly challenging, but the ability to detect production of specific ROS provides important information when interrogating the role of oxidative stress or redox regulated signaling in biological settings. The appropriate handling of tissue when using spin probes and EPR is essential to generate reproducible and meaningful results. The use of spin probes with tissue will not likely measure superoxide radicals present at the time of tissue harvesting due to a short half-life, but instead it detects superoxide produced by enzymes such as NAPDH oxidase, uncoupled endothelial nitric oxide synthase, or xanthine oxidase when lung tissue or homogenates are incubated with the spin probe at 37 °C. The use of frozen tissue will not include superoxide generated by mitochondria, since freezing damages mitochondrial electron transport chain activity. To test mitochondrial superoxide, investigators need to isolate fresh mitochondria or use mitochondrial specific probes in vivo or in fresh tissue.
Several different protocols to preserve tissue have been published in the literature14,15. We compared three published methods for EPR measurements in lung tissue: 1) flash freezing tissue in liquid nitrogen, 2) homogenizing tissue in sucrose buffer, and 3) treating mice in vivo with a spin probe 1 hour before tissue harvesting. We compared control mice to mice with severe lung inflammation and oxidative stress induced by bleomycin to test each method's ability to show consistent differences in nitroxide signals in injured lungs. All three methods showed a similar relative increase in nitroxide signal in the lungs of bleomycin-treated mice. The use of flash frozen tissue would likely be the easiest approach to collect tissue for most labs, negating the need to process tissue in the sucrose buffer at the time of harvesting. The injection of CPH to capture free radicals in vivo is powerful, but to confirm the specific species, this requires a treatment group including the appropriate antioxidant.
One challenge of using spin probes is that the oxidation of spin probes to nitroxide generates a similar three-line EPR spectrum regardless of the species responsible for the oxidation; thus, it does not distinguish between different ROS species. Also, it has been reported that there are potential reactions of hydroxylamine probes with photosynthetic electron transport chain and cytochrome c oxidase19,20. These observations should be considered when interpreting results. In this protocol, the photosynthetic system is not present, and the inclusion of DTPA with the buffer inhibits potential contamination of free ferric and cuprous ions10 . We demonstrated how to use a series of specific enzymes or chelators in lung tissue to establish the contribution of particular ROS or enzyme inhibitors to determine the source of ROS. This approach has been previously used with EPR to determine the contribution of ROS due to uncoupled eNOS13,15. We provide a list of common inhibitors used to distinguish species responsible for spin probe oxidation (Table 1).
We also demonstrated the importance of optimizing the incubation time for each experimental condition. When comparing spin probes to spin traps, spin traps generate unique spectra depending on the reactant which allows for specificity of the free radical species; however, they also exhibit slow kinetics for superoxide spin trapping and are prone to biodegradation. The treatment of lung tissue with the EPR probe ex vivo is also limited by an inability to adequately distinguish extracellular from intracellular superoxide due to the disruption of cell membranes during processing of the tissue (freezing or homogenizing). Use of the injected spin probe in vivo in conjunction with SOD or cell-permeable PEG-SOD can address this problem.
One goal was to establish a protocol to efficiently collect samples and store them at -80 °C prior to EPR measurements. We therefore developed a practical method to use PTFE tubing for holding the samples. This tubing is placed directly into the finger dewar for EPR analysis at 77 K without the need to clean the dewar between samples. This is an alternative to the recently published method involving the freezing of samples in 1 mL syringes. The measurements in frozen samples stored in PTFE tubing can be repeated over several days to demonstrate stability of the signal. This approach allows for batching the EPR measurements and facilitates transferring of the samples between laboratories so a remote EPR facility can run samples.
Overall, these protocols provide a straightforward approach to preparing cells and tissues for EPR measurements in biological systems. The protocols can be adapted to other models associated with oxidative stress and with the use of other spin probes. The timing and concentration of the spin probe will need to be adjusted for each experimental condition. The ability of EPR to determine the presence and production of free radical species unambiguously provides rigor to experimental approaches in the field of redox biology.
The authors have nothing to disclose.
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.
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 |