Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 states^2,3,4,5,6,7. 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 research^{9,10,11,12,13}.

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 models^14,15. 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.

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Protokół

All animal studies were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.

1. Preparation of Reagents

1. Diethylenetriaminepentaacetic acid (DTPA) stock (150 mM)
   1. Add 2.95 g of DTPA (393.35 g/mol) to 10 mL of deionized water.
   2. To dissolve DTPA, add 1 M NaOH dropwise and bring to a pH of 7.0.
   3. Bring the volume to 50 mL with water for a final DTPA concentration of 150 mM, and store at 4 °C.
2. Phosphate buffer saline (PBS) (50 mM, pH 7.4)
   1. Prepare 5 M of sodium chloride (NaCl) (58.44 g/mol; 29.22 g/100 mL).
   2. Prepare 1 M of potassium phosphate dibasic HK₂PO₄ (174.18 g/mol; 17.42 g/100 mL)
   3. Prepare 1 M of potassium phosphate monobasic KH₂PO₄ (136.1 g/mol; 13.61 g/100 mL). Mix 3 mL of 5 M NaCl with 4.24 mL of 1 M potassium phosphate dibasic and 0.760 mL of 1 M potassium phosphate monobasic. Check the pH.
   4. Bring the volume to 100 mL with deionized water.
   5. Store at room temperature (RT) for short-term (days) and at 4 °C for long-term (weeks) storage.
3. Krebs-Henseleit buffer (KHB) containing 100 µM DTPA
   1. In 50 mL conical centrifuge tube, add 33.3 µL of 150 mM DTPA stock solution.
   2. Bring to a 50 mL volume with Krebs-Henseleit buffer (KHB).
   3. Prepare fresh buffer with DTPA each day and keep it at RT.
4. Tris-EDTA buffer containing sucrose
   1. Prepare 0.5 M Tris stock: dissolve 15.14 g of Tris base (121.14 g/mol) in 150 mL of deionized water. Using HCl, adjust the pH to 7.8 and bring to a final volume of 250 mL.
   2. Dissolve 21.4 g of sucrose (342.29 g/mol; final concentration = 0.25 mM) in 150 mL of deionized water.
   3. Add 5 mL of Tris stock to sucrose to achieve a 10 mM final Tris concentration.
   4. Add 0.5 mL of 0.5 M EDTA stock to Tris-sucrose to achieve a 1 mM final concentration.
   5. Check the pH and adjust it to 7.4.
   6. Bring to a final volume of 250 mL with deionized water and store at 4 °C.
5. Bovine erythrocyte Cu/Zn superoxide dismutase (SOD) stock (30,000 U/mL)
   1. Reconstitute 30,000 U of SOD in 1 mL of PBS (approximately 5.7 mg, depending on activity of SOD lot).
   2. Mix well, aliquot, and store at -20° C for short-term (6-12 months) and at -80 °C for long-term storage.
6. SOD working solution (1000 U/mL)
   1. Transfer a 30 µL aliquot of 30,000 U/mL SOD stock into a  870 µL of sterile PBS.
   2. Keep the solution on ice, and use it fresh.
7. Phorbol 12-myristate 13-acetate (PMA) stock (5 mM)
   1. Dissolve 1 mg of PMA (616.83 g/mol) in 325 µL of DMSO (final concentration = 5 mM).
   2. Aliquot a 5 mM PMA solution and store it at -20 °C.
8. PMA working solution (125 µM)
   1. Dilute a 10 µL aliquot of 5 mM PMA stock into 390 µL of sterile PBS.
   2. Keep the solution on ice and use it fresh.
   3. For a vehicle control for PMA, use 10 µL of DMSO in 390 µL of PBS.
9. Diphenyliodonium chloride (DIP) (2.5 mM)
   1. Dissolve 3.2 mg of DIP (316.57 g/mol) in 4 mL to obtain a 2.5 mM stock.
   2. Prepare the solution and use it fresh.
10. Deferoxamine mesylate salt (DFO) (20 mM)
    1. Dissolve 4.5 mg of DFO (656.79 g/ mol) in 340 µL to obtain a 20 mM stock.
    2. Prepare the solution and use it fresh.
11. Preparation of antimycin A (AA) stock (5 mM)
    1. Dissolve 5.4 mg of AA (532 g/mol) in 2 mL of ethanol (final concentration = 5 mM).
    2. Aliquot the stock in glass vials and store at -20 °C.
12. Preparation of spin probes
    1. Bubble 50 mM phosphate buffer containing 100 µM DTPA with nitrogen for 30 min to remove dissolved oxygen from the buffer.
    2. Remove the spin probe from the -20 °C freezer and allow the container to come to RT (10-15 min).
    3. Weigh out 2.4 mg of 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl (CMH) (237.8 g/mol)
    4. Dissolve CMH into 1 mL of the deoxygenated phosphate buffer for a final concentration of 10 mM.
    5. Weigh out 5 mg of 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) (529.1 g/mol).
    6. Dissolve mito-TEMPO-H into 1 mL of the deoxygenated phosphate buffer for a final concentration of 9.5 mM.
    7. Weigh out 4.9 mg of 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine·HCl (CPH) (223.7 g/mol).
    8. Dissolve CPH into 1 mL of the deoxygenated phosphate buffer for a final concentration of 22 mM.
    9. Aliquot and store at -80 °C (freeze-thaw is not recommended).

2. Detection of Superoxide in vitro

1. Detection of total, extracellular, and intracellular superoxide in PMA-stimulated RAW 264.7 cells at RT
   1. Following proper aseptic technique, thaw RAW 264.7 cells and passage them in DMEM media supplemented with 10% FBS (low endotoxin-free) and 1% antimycotic/ampicillin at 37 °C in CO₂ incubator.
   2. Seed RAW 264.7 cells at 1 x 10⁶ cells/well into 6-well plates one day prior to treatment.
   3. Gently remove media and wash the cells once with 1 mL of KHB buffer.
   4. Add KHB containing 100 µM DTPA to each well, and treat in a total volume of 500 µL with the following:
   5. For wells pretreated with SOD, add 15 µL/well of SOD working solution (1000 U/mL; final concentration of SOD = 30 U/mL) and incubate for 10 min at 37 °C prior to addition of CMH and PMA.
   6. Add 12.5 µL/well of 10 mM CMH stock (final concentration = 0.25 mM).
   7. Add 40 µL/well of 125 µM PMA working solution (final concentration = 10 µM) or 40 µL vehicle (stock 10 µL of DMSO in 390 µL of PBS).
   8. Incubate for 50 min at 37 °C in a CO₂ incubator.
   9. Remove the plates from the incubator and place them immediately on ice.
   10. Collect buffer from each well in separate, 1.5 mL, labeled tubes. Keep on ice throughout.
   11. Add 100 µL of fresh KHB buffer containing 100 µM DTPA, gently scrape the cells, and resuspend by pipetting up and down several times. Keep on ice throughout cell resuspension.
   12. Load the sample collected in steps 2.1.10 and 2.1.11 (50 µL) in each of the capillary tubes. Seal both ends and run the EPR.
       NOTE: Always test a tube or well (without cells) containing the probe in buffer (same concentration = 0.25 mM), treated under the same conditions as the cells (same incubation time and temperature) as a control, since background intensity of the probe is temperature- and time-dependent.
   13. Set the EPR acquisition parameters to the following: microwave frequency = 9.65 GHz; center field = 3432 G; modulation amplitude = 2.0 G; sweep width = 80 G; microwave power = 19.9 mW; total number of scans = 10; sweep time = 12.11 s; and time constant = 20.48 ms.
2. Detection of mitochondrial superoxide in RAW 264.7 cells
   1. Follow steps 2.1.1 and 2.1.2 to seed RAW 264.7 cells one day prior to the experiment.
   2. Remove media and wash the cells once with 1 mL of KHB buffer.
   3. Add 200 µL of KHB containing 100 µM DTPA to each well.
   4. Add 5.3 µL/well of 9.5 mM mito-TEMPO-H stock (final concentration = 0.25 mM)
   5. Incubate for 10 min at RT.
   6. Add 1 µL/well of antimycin A (AA), 5 mM stock solution in ethanol (final concentration = 25 µM).
   7. Incubate for 50 min at 37 °C in a CO₂ incubator.
   8. Remove the plates from the incubator and place them immediately on ice.
   9. Gently scrape the cells and resuspend by pipetting up and down. Keep on ice.
   10. Load the sample in a capillary tube. Seal both ends.
   11. See the previous section for EPR setting.
3. Detection of superoxide in RAW 264.7 cells at 77 K
   1. Place the buffer collected in step 1.1.10 in pre-prepared PTFE tubing 1-2 inches in length (3/16" OD x 1/8" ID). Make sure the PTFE tubing is straight so it can be easily inserted and removed from the finger dewar. Use a rubber stopper to close one end of the PTFE tubing, pipette the buffer or cell suspension (100 to 150 µL) into the PTFE tubing, and seal the tubing with a second stopper.
   2. Flash freeze the sample in liquid nitrogen. The sample can be transferred to a labeled cryopreservation tube for storage at -80 °C or run immediately.
   3. Fill the finger dewar with liquid nitrogen and insert the PTFE tubing containing the sample into the finger dewar. Make sure the sample is centered in the active space of the resonator and run EPR at 77 K.
      NOTE: Start the nitrogen gas flow to your spectrometer 15-30 min before the measurements, and continue this flow throughout the measurements to prevent water condensation in the resonator.
   4. Set EPR acquisition parameters to the following: microwave frequency = 9.65 GHz; center field = 3438 G; modulation amplitude = 4.0 G; sweep width = 150 G; microwave power = 0.316 mW; total number of scans = 10; sweep time = 60 s; and time constant = 1.28 ms.

3. EPR Measurements in Fluids

1. Whole blood
   1. Treat mice (8-12 weeks old) with a single dose of intratracheal bleomycin (Bleo; 100 µL at 1 U/mL) dissolved in PBS or PBS alone as previously described^16,17.
   2. Euthanize mice by administering inhaled isoflurane (1.5-4%) followed by exsanguination and cervical dislocation. Aspirate blood through the right ventricle into a syringe coated with heparin (1000 USP/mL) containing 100 µM DTPA and transfer to a 1.5 mL tube.
   3. In a separate 1.5 mL tube, add 15 µL of PBS containing 100 µM DTPA and 3 µL of CMH (10 mM) to 132 µL of blood for a total volume of 150 µL and final CMH concentration of 0.2 mM.
   4. Incubate blood for 10 min at 37 °C in a water bath.
   5. Remove the tubes from water bath.
   6. Take an aliquot by loading blood in a capillary tube and run EPR at RT with the following EPR acquisition parameters: microwave frequency = 9.65 GHz; center field = 3432 G; modulation amplitude = 1.0 G; sweep width = 80 G; microwave power = 19.9 mW; total number of scans = 3; sweep time = 12.11 s; and time constant = 20.48 ms. Alternatively, samples can be flash frozen as described in step 2.3 for measurements at 77 K. EPR acquisition parameters are the following: microwave frequency = 9.65 GHz; center field = 3438 G; modulation amplitude = 4.0 G; sweep width = 150 G; microwave power = 0.316 mW; total number of scans = 2; sweep time = 60 s; and time constant = 1.28 ms.
2. Bronchoalveolar lavage fluid (BALF)
   1. After euthanasia (see step 3.1.2), collect BALF by slowly instilling and withdrawing 1 mL of PBS containing 100 µM DTPA three times in a syringe via a cannula placed in the trachea.
   2. In a 1.5 mL tube, treat 200 µL of BALF with 4 µL of CMH (10 mM) to obtain a final concentration of 0.2 mM.
   3. Incubate BALF for 50 min at 37 °C in a water bath.
   4. Take tubes out of the water bath and place them on ice.
   5. Load BALF in a capillary tube and run EPR at RT with the same EPR settings as used in step 1.1.13, or flash freeze in liquid nitrogen as described in step 2.3.
3. EPR measurements in blood and BALF at 77 K
   1. Follow the protocol above to collect blood (steps 3.1.1. to 3.1.4) and BALF (steps 3.2.1 to 3.2.4).
   2. Place 150 µL of the treated blood or BALF in PTFE tubing (1-2 in). Use a rubber stopper to close one end of the PTFE tubing prior to adding the sample and another stopper to seal the tubing.
   3. Flash freeze the sample in liquid nitrogen.
   4. See section 2.3 for details on running EPR in frozen samples in PTFE tubing using the finger dewar at 77 K. Run Frozen CMH treated Blood samples with in a week.

4. EPR Measurements on Lung Tissue

1. Flash frozen lung tissue
   1. After collecting the BALF in step 3.2.1, the chest is opened and lungs flushed with 10 mL of cold PBS via the right ventricle to remove blood. Flash freeze the lung tissue in liquid nitrogen. Frozen lung tissue can be stored at -80 °C for up to 6 months until use for EPR measurements.
   2. Stabilize the lung tissue on dry ice with tweezers and cut multiple small pieces (5-15 mg) of lung tissue using a single-edge blade.
   3. Weigh the tissue in a 1.5 mL tube, place the tube on the scale and tare the scale, then add the tissue pieces and record the weight.
   4. To the tissue in the 1.5 mL tube, add 196 µL of KHB containing DTPA and 4 µL of CMH (0.2 mM) to achieve a 200 µL total volume.
   5. Incubate for 1 h at 37 °C in a water bath.
   6. Spin down (for a few seconds) in a microcentrifuge at 3,884 x g.
   7. Place on ice and pipette 150 µL of the supernatant into the PTFE tubing and freeze for the 77 K measurements as described in section 2.3.
      NOTE: For this method, the heterogeneity of the injury needs to be considered. For a bleomycin-induced lung injury, given that it is a highly heterogeneous injury, it is recommended to cut several tissue pieces from different parts of the lung from each mouse. Alternatively, a larger piece of tissue can be homogenized in KHB buffer containing 100 µM DTPA at a 1:6 weight-to-volume ratio (mg/µL) as described below.
2. Fresh lung tissue preserved in sucrose buffer
   1. Flush the lavaged lungs with cold PBS to remove blood as done in step 3.1.2.
   2. Homogenize the fresh lung tissue in Tris-EDTA buffer containing 0.25 M sucrose with a 1:6 lung/buffer (mg/µL) ratio using Dounce tissue grinder with a glass or PTFE pestle.
   3. Add 50 µL of the lung homogenate to 450 µL of KHB containing 100 µM DTPA.
   4. In a 1.5 mL tube (in a total volume of 100 µL), to 98 µL of lung homogenate in KHB, add 2 µL of CMH of 10 mM stock to obtain a final concentration of 0.2 mM.
   5. Incubate for 20 min 37 °C in a water bath.
   6. Place the samples on ice and load them in a capillary tube. Run EPR at RT (settings used in step 2.1.13).
   7. To test the contribution of specific species and sources using different inhibitors, pre-treat 88 µL of lung homogenate +/- inhibitor, adjusting with KHB to achieve a final volume of 98 µL. In this experiment, the inhibitors included 10 µL of SOD (100 U/mL), 4 µL of deferoxamine (DFO; final concentration = 800 µM), or 4 µL of diphenyliodonium chloride (DIP; final concentration = 100 µM). Incubate for 20 min at 37 °C in a water bath.
   8. Add 2 µL of CMH and incubate for another 20 min at 37 °C, followed by EPR measurements as described above. Include a one-time matched blank sample with CMH KHB containing sucrose buffer. Alternatively, store aliquots of the remaining lung homogenates (step 3.1.2) at -80 °C for future measurements.
      NOTE: The total volume can be scaled as needed.
3. EPR measurements on lung tissue from mice injected with spin probes in vivo (at RT using tissue cell)
   1. Prepare CPH stock solution by dissolving 4.9 mg of CPH in 1 mL of filtered and deoxygenated 50 mM phosphate buffer.
   2. Anesthetize mice with inhaled isoflurane (1.5-4%) for 20-30 seconds until unresponsive to toe pinch. Inject mice via retroorbital route with 100 µL of CPH spin probe for a 25 g mouse body weight (final dose = 20 mg/kg), and allow the probe to circulate for 1 h. Immediately after retroorbital injection, add one drop of 0.5% proparacaine HCl on the eye area to prevent eye pain and dryness. Monitor mice for 1 h and proceed to tissue harvesting.
   3. Harvest the lung tissue as described above and flash freeze the lungs.
   4. Cut 20-30 mg of frozen tissue on dry ice and record the exact weight.
   5. Gently wipe the tissue with cleaning wipes to absorb any surface water.
   6. Place the tissue within the window of the tissue cell (an accessory allows EPR measurements for tissue samples) and run EPR to determine total spins. The data can be expressed as total spins per mg of tissue.

5. Data Analysis

1. Simulate the EPR spectra using SpinFit module incorporated in the Xenon software of the bench-top EMXnano EPR spectrometer. Determine the nitroxide concentration by the SpinCount module. Alternatively, a calibration curve of a stable nitroxide such as 4-hydroxy-TEMPO or TEMPOL can be made, and the concentration can be obtained by comparing the intensity of the signal with the sample and standard.
2. For the data collected at 77 K, use double integration followed by SpinCount.

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Wyniki

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...

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Dyskusje

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 ...

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Materiały

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