This work was supported by the University of Colorado School of Medicine Dean&#39;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,&#160;Ashley Trumpie and Ivy McDermott (University of Colorado Denver) for technical support.

All animal studies were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
1. Preparation of Reagents
<ol>
	<li>Diethylenetriaminepentaacetic acid (DTPA) stock (150 mM)
	<ol>
		<li>Add 2.95 g of DTPA (393.35 g/mol) to 10 mL of deionized water.</li>
		<li>To dissolve DTPA, add 1 M NaOH dropwise and bring to a pH of 7.0.</li>
		<li>Bring the volume to 50 mL with water for a final DTPA concentration of 150 mM, and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Phosphate buffer saline (PBS) (50 mM, pH 7.4)
	<ol>
		<li>Prepare 5 M of sodium chloride (NaCl) (58.44 g/mol; 29.22 g/100 mL).</li>
		<li>Prepare 1 M of potassium phosphate dibasic HK2PO4 (174.18 g/mol; 17.42 g/100 mL)</li>
		<li>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.</li>
		<li>Bring the volume to 100 mL with deionized water.</li>
		<li>Store at room temperature (RT) for short-term (days) and at 4 &#176;C for long-term (weeks) storage.</li>
	</ol>
	</li>
	<li>Krebs-Henseleit buffer (KHB) containing 100 &#181;M DTPA 
	<ol>
		<li>In 50 mL conical centrifuge tube, add 33.3 &#181;L of 150 mM DTPA stock solution.</li>
		<li>Bring to a 50 mL volume with Krebs-Henseleit buffer (KHB).</li>
		<li>Prepare fresh buffer with DTPA each day and keep it at RT.</li>
	</ol>
	</li>
	<li>Tris-EDTA buffer containing sucrose
	<ol>
		<li>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.</li>
		<li>Dissolve 21.4 g of sucrose (342.29 g/mol; final concentration = 0.25 mM) in 150 mL of deionized water.</li>
		<li>Add 5 mL of Tris stock to sucrose to achieve a 10 mM final Tris concentration.</li>
		<li>Add 0.5 mL of 0.5 M EDTA stock to Tris-sucrose to achieve a 1 mM final concentration.</li>
		<li>Check the pH and adjust it to 7.4.</li>
		<li>Bring to a final volume of 250 mL with deionized water and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Bovine erythrocyte Cu/Zn superoxide dismutase (SOD) stock (30,000 U/mL) 
	<ol>
		<li>Reconstitute 30,000 U of SOD in 1 mL of PBS (approximately 5.7 mg, depending on activity of SOD lot).</li>
		<li>Mix well, aliquot, and store at -20&#176; C for short-term (6-12 months) and at -80 &#176;C for long-term storage.</li>
	</ol>
	</li>
	<li>SOD working solution (1000 U/mL)
	<ol>
		<li>Transfer a 30 &#181;L aliquot of 30,000 U/mL SOD stock into a &#160;870 &#181;L of sterile PBS.</li>
		<li>Keep the solution on ice, and use it fresh.</li>
	</ol>
	</li>
	<li>Phorbol 12-myristate 13-acetate (PMA) stock (5 mM)
	<ol>
		<li>Dissolve 1 mg of PMA (616.83 g/mol) in 325 &#181;L of DMSO (final concentration = 5 mM).</li>
		<li>Aliquot a 5 mM PMA solution and store it at -20 &#176;C.</li>
	</ol>
	</li>
	<li>PMA working solution (125 &#181;M)
	<ol>
		<li>Dilute a 10 &#181;L aliquot of 5 mM PMA stock into 390 &#181;L of sterile PBS.</li>
		<li>Keep the solution on ice and use it fresh.</li>
		<li>For a vehicle control for PMA, use 10 &#181;L of DMSO in 390 &#181;L of PBS.</li>
	</ol>
	</li>
	<li>Diphenyliodonium chloride (DIP) (2.5 mM)
	<ol>
		<li>Dissolve 3.2 mg of DIP (316.57 g/mol) in 4 mL to obtain a 2.5 mM stock.</li>
		<li>Prepare the solution and use it fresh.</li>
	</ol>
	</li>
	<li>Deferoxamine mesylate salt (DFO) (20 mM)
	<ol>
		<li>Dissolve 4.5 mg of DFO (656.79 g/ mol) in 340 &#181;L to obtain a 20 mM stock.</li>
		<li>Prepare the solution and use it fresh.</li>
	</ol>
	</li>
	<li>Preparation of antimycin A (AA) stock (5 mM)
	<ol>
		<li>Dissolve 5.4 mg of AA (532 g/mol) in 2 mL of ethanol (final concentration = 5 mM).</li>
		<li>Aliquot the stock in glass vials and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of spin probes 
	<ol>
		<li>Bubble 50 mM phosphate buffer containing 100 &#181;M DTPA with nitrogen for 30 min to remove dissolved oxygen from the buffer.</li>
		<li>Remove the spin probe from the -20 &#176;C freezer and allow the container to come to RT (10-15 min).</li>
		<li>Weigh out 2.4 mg of 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine&#183;HCl (CMH) (237.8 g/mol)</li>
		<li>Dissolve CMH into 1 mL of the deoxygenated phosphate buffer for a final concentration of 10 mM.</li>
		<li>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).</li>
		<li>Dissolve mito-TEMPO-H into 1 mL of the deoxygenated phosphate buffer for a final concentration of 9.5 mM.</li>
		<li>Weigh out 4.9 mg of 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine&#183;HCl (CPH) (223.7 g/mol).</li>
		<li>Dissolve CPH into 1 mL of the deoxygenated phosphate buffer for a final concentration of 22 mM.</li>
		<li>Aliquot and store at -80 &#176;C (freeze-thaw is not recommended).</li>
	</ol>
	</li>
</ol>
2. Detection of Superoxide in vitro
<ol>
	<li>Detection of total, extracellular, and intracellular superoxide in PMA-stimulated RAW 264.7 cells at RT
	<ol>
		<li>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 &#176;C in CO2 incubator.</li>
		<li>Seed RAW 264.7 cells at 1 x 106 cells/well into 6-well plates one day prior to treatment.</li>
		<li>Gently remove media and wash the cells once with 1 mL of KHB buffer.</li>
		<li>Add KHB containing 100 &#181;M DTPA to each well, and treat in a total volume of 500 &#181;L with the following:</li>
		<li>For wells pretreated with SOD, add 15 &#181;L/well of SOD working solution (1000 U/mL; final concentration of SOD = 30 U/mL) and incubate for 10 min at 37 &#176;C prior to addition of CMH and PMA.</li>
		<li>Add 12.5 &#181;L/well of 10 mM CMH stock (final concentration = 0.25 mM).</li>
		<li>Add 40 &#181;L/well of 125 &#181;M PMA working solution (final concentration = 10 &#181;M) or 40 &#181;L vehicle (stock 10 &#181;L of DMSO in 390 &#181;L of PBS).</li>
		<li>Incubate for 50 min at 37 &#176;C in a CO2 incubator.</li>
		<li>Remove the plates from the incubator and place them immediately on ice.</li>
		<li>Collect buffer from each well in separate, 1.5 mL, labeled tubes. Keep on ice throughout.</li>
		<li>Add 100 &#181;L of fresh KHB buffer containing 100 &#181;M DTPA, gently scrape the cells, and resuspend by pipetting up and down several times. Keep on ice throughout cell resuspension.</li>
		<li>Load the sample collected in steps 2.1.10 and 2.1.11 (50 &#181;L) 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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Detection of mitochondrial superoxide in RAW 264.7 cells
	<ol>
		<li>Follow steps 2.1.1 and 2.1.2 to seed RAW 264.7 cells one day prior to the experiment.</li>
		<li>Remove media and wash the cells once with 1 mL of KHB buffer.</li>
		<li>Add 200 &#181;L of KHB containing 100 &#181;M DTPA to each well.</li>
		<li>Add 5.3 &#181;L/well of 9.5 mM mito-TEMPO-H stock (final concentration = 0.25 mM)</li>
		<li>Incubate for 10 min at RT.</li>
		<li>Add 1 &#181;L/well of antimycin A (AA), 5 mM stock solution in ethanol (final concentration = 25 &#181;M).</li>
		<li>Incubate for 50 min at 37 &#176;C in a CO2 incubator.</li>
		<li>Remove the plates from the incubator and place them immediately on ice.</li>
		<li>Gently scrape the cells and resuspend by pipetting up and down. Keep on ice.</li>
		<li>Load the sample in a capillary tube. Seal both ends.</li>
		<li>See the previous section for EPR setting.</li>
	</ol>
	</li>
	<li>Detection of superoxide in RAW 264.7 cells at 77 K
	<ol>
		<li>Place the buffer collected in step 1.1.10 in pre-prepared PTFE tubing 1-2 inches in length (3/16&#34; OD x 1/8&#34; 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 &#181;L) into the PTFE tubing, and seal the tubing with a second stopper.</li>
		<li>Flash freeze the sample in liquid nitrogen. The sample can be transferred to a labeled cryopreservation tube for storage at -80 &#176;C or run immediately.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. EPR Measurements in Fluids
<ol>
	<li>Whole blood 
	<ol>
		<li>Treat mice (8-12 weeks old) with a single dose of intratracheal bleomycin (Bleo; 100 &#181;L at 1 U/mL) dissolved in PBS or PBS alone as previously described16,17.</li>
		<li>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 &#181;M DTPA and transfer to a 1.5 mL tube.</li>
		<li>In a separate 1.5 mL tube, add 15 &#181;L of PBS containing 100 &#181;M DTPA and 3 &#181;L of CMH (10 mM) to 132 &#181;L of blood for a total volume of 150 &#181;L and final CMH concentration of 0.2 mM.</li>
		<li>Incubate blood for 10 min at 37 &#176;C in a water bath.</li>
		<li>Remove the tubes from water bath.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Bronchoalveolar lavage fluid (BALF)
	<ol>
		<li>After euthanasia (see step 3.1.2), collect BALF by slowly instilling and withdrawing 1 mL of PBS containing 100 &#181;M DTPA three times in a syringe via a cannula placed in the trachea.</li>
		<li>In a 1.5 mL tube, treat 200 &#181;L of BALF with 4 &#181;L of CMH (10 mM) to obtain a final concentration of 0.2 mM.</li>
		<li>Incubate BALF for 50 min at 37 &#176;C in a water bath.</li>
		<li>Take tubes out of the water bath and place them on ice.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>EPR measurements in blood and BALF at 77 K
	<ol>
		<li>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).</li>
		<li>Place 150 &#181;L 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.</li>
		<li>Flash freeze the sample in liquid nitrogen.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. EPR Measurements on Lung Tissue
<ol>
	<li>Flash frozen lung tissue 
	<ol>
		<li>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 &#176;C for up to 6 months until use for EPR measurements.</li>
		<li>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.</li>
		<li>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.</li>
		<li>To the tissue in the 1.5 mL tube, add 196 &#181;L of KHB containing DTPA and 4 &#181;L of CMH (0.2 mM) to achieve a 200 &#181;L total volume.</li>
		<li>Incubate for 1 h at 37 &#176;C in a water bath.</li>
		<li>Spin down (for a few seconds) in a microcentrifuge at 3,884 x g.</li>
		<li>Place on ice and pipette 150 &#181;L 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 &#181;M DTPA at a 1:6 weight-to-volume ratio (mg/&#181;L) as described below.</li>
	</ol>
	</li>
	<li>Fresh lung tissue preserved in sucrose buffer 
	<ol>
		<li>Flush the lavaged lungs with cold PBS to remove blood as done in step 3.1.2.</li>
		<li>Homogenize the fresh lung tissue in Tris-EDTA buffer containing 0.25 M sucrose with a 1:6 lung/buffer (mg/&#181;L) ratio using Dounce tissue grinder with a glass or PTFE pestle.</li>
		<li>Add 50 &#181;L of the lung homogenate to 450 &#181;L of KHB containing 100 &#181;M DTPA.</li>
		<li>In a 1.5 mL tube (in a total volume of 100 &#181;L), to 98 &#181;L of lung homogenate in KHB, add 2 &#181;L of CMH of 10 mM stock to obtain a final concentration of 0.2 mM.</li>
		<li>Incubate for 20 min 37 &#176;C in a water bath.</li>
		<li>Place the samples on ice and load them in a capillary tube. Run EPR at RT (settings used in step 2.1.13).</li>
		<li>To test the contribution of specific species and sources using different inhibitors, pre-treat 88 &#181;L of lung homogenate +/- inhibitor, adjusting with KHB to achieve a final volume of 98 &#181;L. In this experiment, the inhibitors included 10 &#181;L of SOD (100 U/mL), 4 &#181;L of deferoxamine (DFO; final concentration = 800 &#181;M), or 4 &#181;L of diphenyliodonium chloride (DIP; final concentration = 100 &#181;M). Incubate for 20 min at 37 &#176;C in a water bath.</li>
		<li>Add 2 &#181;L of CMH and incubate for another 20 min at 37 &#176;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 &#176;C for future measurements. 
		NOTE: The total volume can be scaled as needed.</li>
	</ol>
	</li>
	<li>EPR measurements on lung tissue from mice injected with spin probes in vivo (at RT using tissue cell)
	<ol>
		<li>Prepare CPH stock solution by dissolving 4.9 mg of CPH in 1 mL of filtered and deoxygenated 50 mM phosphate buffer.</li>
		<li>Anesthetize mice with inhaled isoflurane (1.5-4%) for 20-30 seconds until unresponsive to toe pinch. Inject mice via retroorbital route with 100 &#181;L 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.</li>
		<li>Harvest the lung tissue as described above and flash freeze the lungs.</li>
		<li>Cut 20-30 mg of frozen tissue on dry ice and record the exact weight.</li>
		<li>Gently wipe the tissue with cleaning wipes to absorb any surface water.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Data Analysis
<ol>
	<li>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.</li>
	<li>For the data collected at 77 K, use double integration followed by SpinCount.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Targeting+of+Mitochondrial+Superoxide+in+Hypertension.">Therapeutic Targeting of Mitochondrial Superoxide in Hypertension.</a> Circulation Research. 107 (1), 106-116 (2010).</li><li>Dikalov, S. I., Polienko, Y. F., Kirilyuk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Paramagnetic+Resonance+Measurements+of+Reactive+Oxygen+Species+by+Cyclic+Hydroxylamine+Spin+Probes.">Electron Paramagnetic Resonance Measurements of Reactive Oxygen Species by Cyclic Hydroxylamine Spin Probes.</a> Antioxidants &amp; Redox Signaling. , (2017).</li><li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Carnitine+preserves+endothelial+function+in+a+lamb+model+of+increased+pulmonary+blood+flow.">L-Carnitine preserves endothelial function in a lamb model of increased pulmonary blood flow.</a> Pediatric Research. 74 (1), 39-47 (2013).</li><li>Berg, K., Ericsson, M., Lindgren, M., Gustafsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High+Precision+Method+for+Quantitative+Measurements+of+Reactive+Oxygen+Species+in+Frozen+Biopsies.">A High Precision Method for Quantitative Measurements of Reactive Oxygen Species in Frozen Biopsies.</a> PloS One. 9 (3), (2014).</li><li>Kozlov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EPR+analysis+reveals+three+tissues+responding+to+endotoxin+by+increased+formation+of+reactive+oxygen+and+nitrogen+species.">EPR analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species.</a> Free Radical Biology and Medicine. 34 (12), 1555-1562 (2003).</li><li>Van Rheen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Extracellular+Superoxide+Dismutase+Overexpression+Lessens+Bleomycin-Induced+Pulmonary+Hypertension+and+Vascular+Remodeling.">Lung Extracellular Superoxide Dismutase Overexpression Lessens Bleomycin-Induced Pulmonary Hypertension and Vascular Remodeling.</a> American Journal of Respiratory Cell and Molecular Biology. 44 (4), 500-508 (2011).</li><li>Mouradian, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+Dismutase+3+R213G+Single-Nucleotide+Polymorphism+Blocks+Murine+Bleomycin-Induced+Fibrosis+and+Promotes+Resolution+of+Inflammation.">Superoxide Dismutase 3 R213G Single-Nucleotide Polymorphism Blocks Murine Bleomycin-Induced Fibrosis and Promotes Resolution of Inflammation.</a> American Journal of Respiratory Cell and Molecular Biology. 56 (3), 362-371 (2017).</li><li>Dikalov, S. I., Li, W., Mehranpour, P., Wang, S. S., Zafari, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+extracellular+superoxide+by+human+lymphoblast+cell+lines:+comparison+of+electron+spin+resonance+techniques+and+cytochrome+C+reduction+assay.">Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome C reduction assay.</a> Biochem Pharmacol. 73 (7), 972-980 (2007).</li><li>Kozuleva, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+superoxide+radical+production+in+thylakoid+membrane+using+cyclic+hydroxylamines.">Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines.</a> Free Radical Biology and Medicine. 89, 1014-1023 (2015).</li><li>Chen, K., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+Hydroxylamines+to+Nitroxide+Spin+Labels+in+Living+Cells.">Oxidation of Hydroxylamines to Nitroxide Spin Labels in Living Cells.</a> Biochimica Et Biophysica Acta. 970 (3), 270-277 (1988).</li></ol>

The authors have nothing to disclose.

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 ...

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.

<table><tbody><tr><td>DMEM</td><td>LifeTech</td><td>10566-016</td><td>cell culture media</td></tr><tr><td>Diethylenetriaminepentaacetic acid (DTPA)</td><td>Sigma Aldrich</td><td>D6518-5G</td><td></td></tr><tr><td>sodium chloride (NaCl)&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>&amp;nbsp;BP358-212</td><td>used to prepare 50 mM phosphate saline buffer&amp;nbsp; according to Sigma aldrish&amp;nbsp;&amp;nbsp;</td></tr><tr><td>potassium phosphate dibasic (HK&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; )</td><td>Fisher Scientific&amp;nbsp;</td><td>&amp;nbsp;BP363-500</td><td>used to prepare 50 mM phosphate saline buffer&amp;nbsp; according to Sigma aldrish&amp;nbsp;&amp;nbsp;</td></tr><tr><td>potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; )</td><td>Sigma Aldrich</td><td>P-5379</td><td>used to prepare 50 mM phosphate saline buffer&amp;nbsp; according to Sigma aldrish&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Krebs-Henseleit buffer (KHB)&amp;nbsp;</td><td>(Alfa Aesar, Hill)</td><td>J67820</td><td></td></tr><tr><td>Bovine erythrocyte superoxide dismutase (SOD)</td><td>Sigma Aldrich</td><td>&amp;nbsp;S7571-30KU</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Sigma Aldrich</td><td>P1585-1MG</td><td>Dissolve in DMSO</td></tr><tr><td>Antimycin A (AA)</td><td>Sigma Aldrich</td><td>A8674-25MG</td><td>Dissolve in Ethanol and store in glass vials(MW used is the averaged molecular weights for four lots)</td></tr><tr><td>1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine . HCl (CMH)</td><td>Enzo Life Sciences</td><td>ALX-430-117-M050</td><td></td></tr><tr><td>1-Hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine . HCl (CPH)</td><td>Enzo Life Sciences</td><td>ALX-430-078-M250</td><td></td></tr><tr><td>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)</td><td>Enzo Life Sciences</td><td>ALX-430-171-M005</td><td></td></tr><tr><td>1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium chloride . HCl (CAT1H)</td><td>Enzo Life Sciences</td><td>ALX-430-131-M250</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sagent Pharmaceuticals</td><td>NDC 25021-400-10</td><td></td></tr><tr><td>Diphenyliodonium chloride&amp;nbsp;</td><td>Sigma Aldrich</td><td>43088</td><td></td></tr><tr><td>Deferoxamin mesylate salt</td><td>Sigma Aldrich</td><td>D9533-1G</td><td></td></tr><tr><td>Critoseal</td><td>Leica</td><td>39215003</td><td></td></tr><tr><td>BRAND disposable BLAUBRAND&amp;nbsp;micropipettes, intraMark</td><td>Sigma Aldrich</td><td>708733</td><td>Capillaries</td></tr><tr><td>PTFE FRACTIONAL FLUOROPOLYMER TUBING&lt;br/&gt; 3/16&amp;rdquo; OD x 1/8&amp;rdquo; ID</td><td>NORELL</td><td>1598774A</td><td>Teflon tubing&amp;nbsp;</td></tr><tr><td>SILICONE RUBBER STOPPERS FOR NMR SAMPLE TUBES&amp;nbsp; FOR THIN WALL TUBES HAVING AN OD OF 4mm-5mm (3.2mm TO 4.2mm ID) TS-4-5-SR</td><td>NORELL</td><td>94987</td><td></td></tr><tr><td>EMXnano Bench-Top EPR spectrometer&amp;nbsp;</td><td>Bruker BioSpin GmbH</td><td>E7004002</td><td></td></tr><tr><td>EMX NANO TISSUE CELL</td><td>Bruker BioSpin GmbH</td><td>E7004542</td><td></td></tr></tbody></table>

use of electron paramagnetic resonance in biological samples at ambient temperature and 77 k

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,&#160;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 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

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&#160;(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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Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

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 ...

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9.0K Views.  University of Colorado Anschutz Medical Campus. 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.

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

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Chemistry

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Bioengineering

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There exists a drive to characterize materials under conditions relevant to the chemical process of interest. To address this, in situ high-temperature, high-pressure MAS NMR methods have been developed to enable the observation of chemical interactions over a range of pressures (vacuum to several hundred bar) and temperatures (well below 0 &#176;C to 250 &#176;C). Further, the chemical identity of the samples can be comprised of solids, liquids, and gases or mixtures of the three. The method incorporates all-zirconia NMR rotors (sample holder for MAS NMR) which can be sealed using a threaded cap to compress an O-ring. This rotor exhibits great chemical resistance, temperature compatibility, low NMR background, and can withstand high pressures. These combined factors enable it to be utilized in a wide range of system combinations, which in turn permit its use in diverse fields as carbon sequestration, catalysis, material science, geochemistry, and biology. The flexibility of this technique makes it an attractive option for scientists from numerous disciplines.

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

Nuclear magnetic resonance (NMR) spectroscopy represents an important technique to understand the structure and bonding environments of molecules. There ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscience

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Biology

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Electron Paramagnetic Resonance (EPR) Spectroscopy

Source: David C. Powers, Tamara M. Powers, Texas A&#38;M
In this video, we will learn the basic principles behind Electron Paramagnetic Resonance (EPR). We will use EPR spectroscopy to study how dibutylhydroxy toluene (BHT) behaves as an antioxidant in the autoxidation of aliphatic aldehydes.

EPR Spectroscopy, Paramagnetic Molecule and Zeeman Effect

Source: David C. Powers, Tamara M. Powers, Texas A&#38;M
In this video, we will learn the basic principles behind Electron Paramagnetic Resonance (EPR). ...

JoVE Science Education

Inorganic Chemistry

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Source: Winkelaar, G., et al. <a href="https://app.jove.com/v/58537">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> J. Vis. Exp. (2018).
This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale ...

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

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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Electron Paramagnetic Resonance (EPR) Spectroscopy

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions