​Geroscience Redox Biology Core Services

​Redox Status​

​The levels of GSH/GSSG, NADPH/NADP+, NADH/NAD+ will be measured using reverse phase HPLC and quantified using electrochemical, fluorescence, or UV/VIS detection. In addition, in vivo detection of free radicals can be assessed by free radical-targeted MRI.

Changes in redox status can have a major impact on cellular and physiologic processes and are believed to underlie many functional decrements associated with the aging process. Cellular energy production and survival depends upon a series of oxidation/reduction reactions. These reactions can give rise to free radical and pro-oxidant species that can act as regulatory molecules and induce oxidative damage. Thus, the redox potential of a cell is a reflection of multiple interacting molecules and biological processes that influence both oxidant production and removal and ultimately cellular homeostasis.

We have developed high throughput methods for analysis of redox couples using 35 mg tissue per assay as summarized below. The types of samples amenable to analyses include flash frozen tissue, tissue homogenates, cell pellets, and isolated organelles from invertebrates and vertebrates. Samples are extracted with 5% meta-phosphoric acid (for GSH, GSSG) or 125 mM KOH (for NADPH, NADH, NADP+, NAD+). Compounds are resolved by ion pairing reverse phase HPLC and quantified using electrochemical, fluorescence, or UV/VIS detection. The identities of specific compounds have been confirmed by GC-MS and routine spiking of experimental samples with known quantities of standards is utilized to ensure accurate peak assignment. The limits of detection for each metabolite are in the pmol range.


Glutathione Status: Glutathione peroxidase isoforms consume H2O2 upon oxidation of GSH to GSSG. The ratio of GSH to GSSG, therefore, provides a measure of redox status and antioxidant capacity (Puente et al., 2014).


NADPH/NADP+: Regeneration of GSH and reduced thioredoxin, necessary for continued H2O2 consumption by glutathione peroxidases and peroxiredoxins, respectively requires oxidation of NADPH to NADP+. Thus, the relative levels of NADPH and NADP+ provide an additional index of antioxidant capacity.


NADH/NAD+: The ratio of NADH to NAD+ is intimately linked to the redox couples GSH/GSSG and NADPH/NADP+. NADH is the primary carrier of electrons derived from the oxidation of glucose and fatty acids, and the relative ratio of NADH to NAD+ is a determinant of free radical production. In addition, NADH and NADPH can be interconverted by the nicotinamide nucleotide transhydrogenase or NAD+ and NADP+-dependent isoforms of isocitrate dehydrogenase. NAD,NADH, NADP and NADPH are measured as previously described (DeBalsi et al., 2014).


References:


Paul M. Rindler, Angela Cacciola, Michael Kinter, and Luke I. Szweda.  Am J Physiol Heart Circ Physiol.  2016 Nov 1; 311(5): H1091-H1096.


Puente, BN; Kimura, W; Muralidhar, SA; Moon, J; Amatruda, JF; Phelps, KL; Grinsfelder, D; Rothermel, BA; Chen, R; Garcia, JA; Santos, CX; Thet, S; Mori, E; Kinter, M; Rindler, PM; Zacchigna, S; Mukherjee, S; Chen, DJ; Mahmoud, AI; Giacca, M; Rabinovitch, PS; Aroumougame, A; Shah, AM; Szweda, LI; Sadek, HA (2014) The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response. Cell, 157, 565-579.

Tissue and cells must be rapidly frozen and pulverized in liquid nitrogen and shipped on dry ice. It is critical to avoid ischemic periods either during euthanasia or tissue isolation. Ischemia can also change the relative ratio of various redox couples preventing accurate assessment of in vivo steady state concentrations. It is therefore important to euthanize by means that prevents loss of blood flow (e.g. CO2) and to flash freeze tissue for shipment.

​Assay 1:  NADH, & NADPH: $30/sample

Assay 2:  NAD, NADP: ​$30/sample

Assay 3:  GSH & GSSG: $30/sample


Assay 1 & 2: $50/sample

Assays 1, 2, & 3: $75/sample

​Oxidative Damage: Oxidative damage in lipids, DNA, and proteins will be measured by the levels of:

Oxidative Damage (Protein, Lipid, DNA)

F2-isoprostanes (Lipid Peroxidation)

F2-isoprostanes are formed in membranes as a result of free radical attack on arachidonic acid in membrane phospholipids. F2-isoprostanes are chemically stable end-products of lipid peroxidation and reliable and sensitive markers of lipid peroxidation (Morrow and Roberts, 1999; Roberts and Morrow, 2000; Roberts and Morrow, 1995). Because isoprostanes are produced in every tissue, plasma levels of free F2-isoprostanes provide a measure of endogenous production of F2-isoprostanes from all sites in the body, thus providing an excellent marker of whole body oxidative stress levels. Traditional measures of lipid peroxidation, e.g. TBARS or MDA, are unstable and, therefore, difficult to accurately measure. The discovery of F2-isoprostanes and neuroprostanes as a stable and sensitive marker of lipid peroxidation has been a major improvement over other assays of lipid peroxidation. The sample preparation for the isoprostane assay is critical and in the case of isoprostanes is also very labor intensive. The isoprostane level can be measured in frozen samples of 100 mg of tissue, 1 ml of plasma or 300 μl of urine.


References:


Morrow, J.D., Chen, Y., Brame, C.J., Yang, J., Sanchez, S.C., Xu, J., Zackert, W.E., Awad, J.A. and Roberts II, L.J. (1999). The isoprostanes: unique prostaglandin-like products of free-radical-initiated lipid peroxidation. Drug Metab. Rev. 31, 117-139.


Morrow, J.D. and Roberts II, L.J. (1997). The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36, 1-21.


Roberts II, L.J., Morrow, J.D. (2000). Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Rad. Biol. Med. 28, 505-513.


Ward, W.F., Qi, W., Van Remmen, H., Zackett, W.E., Roberts II, L.J., and Richardson, A. (2005). Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels. J. Gerontol. A Biol. Sci. Med. Sci.  60, 847-851.

The levels of F2-isoprostanes (8-iso-PGF2α) are measured using the GC/MS procedure developed by Roberts and Morrow (2000) and described by our group (Ward et al., 2005).


F2-isoprostanes and isofurans will be measured in tissues using gas chromatography-mass spectrometry methods as previously described (Morrow and Roberts, 1994). The isoprostane/isofuran level can be measured in samples of 100 mg of tissue, 1 ml of plasma (requires sample collection from 3-4 mice or 1 rat) or 300 ul of urine. Tissue is homogenized in chloroform:methanol containing BHT (0.005%) to prevent auto-oxidation, dried under a stream of nitrogen, and re-suspended in methanol containing BHT. Esterified F2-isoprostanes (and F2-isofuranes) in phospholipids are saponified by adding aqueous potassium hydroxide (this frees fatty acids from lipids). The sample is acidified and diluted with water. Deuterated-F2-isoprostane internal standard is then added to the mixture. For the measurement of free F2-isoprotanes/F2-isofuranes in plasma, the extraction and hydrolysis steps are omitted, and the sample is simply acidified, diluted, and the internal standard added. The mixture is subsequently run on a silica column to separate isoprostanes/isofuranes from bulk fatty acids. The eluate is converted to pentafluorobenzyl esters, by treatment with pentafluorobenzyl bromide (this step is necessary because free fatty acids are difficult to separate by gas chromatography). The mixture is subjected to thin layer chromatography to remove the excess pentafluorobenzyl bromide and unreacted fatty acids.  The F2-isoprotane/isofurane fraction is extracted using ethyl acetate, and will be analyzed by injection into a Thermo Finnigan TRACE DSQ single quadrupole mass spectrometer. This instrument is capable of electron impact and chemical ionization, with positive and negative ion detection.  The F2-isoprostanes and isofurans are quantified by peak height, and the data is corrected with the internal standard and expressed as nanogram of F2-isoprostanes and F2-isofuranes per mL of plasma or per gram tissue.


References:


Roberts II, L.J., Morrow, J.D. (2000). Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Rad. Biol. Med. 28, 505-513.


Ward, W.F., Qi, W., Van Remmen, H., Zackett, W.E., Roberts II, L.J., and Richardson, A. (2005). Effects of age and caloric restriction on lipid peroxidation: measurement of oxidative stress by F2-isoprostane levels. J. Gerontol. A Biol. Sci. Med. Sci.  60, 847-851.


Jon A. Detterich, Honglei Liu, Silvie Suriany, Roberta M. Kato, Patjanaporn Chalacheva, Bruke Tedla, Payal M. Shah, Michael C. Khoo, John C. Wood, Thomas D. Coates, Ginger L. Milne, Joo-Yeun Oh, Rakesh P.Patel, Henry JayForman (2019). Erythrocyte and plasma oxidative stress appears to be compensated in patients with sickle cell disease during a period of relative health, despite the presence of known oxidative agents. Free Rad. Biol. Med. 141, 408-415

  • Tissue Samples: We require snap frozen tissue stored at -80º C for the F2-isoprostane assay.  For most tissues, 100-150 mg of tissue is optimal for analysis.
  • Cells:  We a need a cell pellet harvested from 2-4 million washed in PBS, frozen in liquid nitrogen and stored at -80º C.
  • Blood:  We require ~1 ml of blood/plasma for the F2-isoprostane assay.  We routinely collect blood from the inferior vena cava of anesthetized animals into pre-chilled heparin-coated tubes. The tubes are centrifuged at 1,500 x g for 10 min at 4º C to give plasma, which is flash-frozen in liquid nitrogen and stored at -80º C.  Normally, enough blood can be collected from one rat for the F2-isoprostane assay; however, for mice blood needs to be pooled from 2 to 4 mice to have enough sample for optimal analysis.

F2-isoprostanes: $125/sample

​8-oxo-deoxyguanosine

For measurement of DNA oxidation (oxo8dG), the major oxidative lesion in DNA, the Core will continue to employ a highly sensitive and reproducible high performance liquid chromatography system with electrochemical detection (HPLC-EC) based assay. A key characteristic of the method we are using is the isolation of DNA using NaI rather than the historically used phenol-based method. We demonstrated that using NaI for isolation of DNA prevents oxidative damage from occurring during sample preparation and the isolation process (Hamilton et al., 2001a). Using this isolation method, we have shown that oxidative damage to DNA (oxo8dG) increases with age in all tissues and strains of mice and rats studied (Hamilton et al., 2001b). The data are expressed as the ratio of nmoles of oxo8dG to 105 nmoles of 2dG.

Detailed methods are found in our previous publications (Hamilton et al., 2001a; Hamilton et al., 2001b).


References:


Hamilton, M.L., Guo, Z.M., Fuller, C.D., Van Remmen, H., Ward, W.F., Austad, S.N., Troyer, D.A., Thompson, I., and Richardson,  A. (2001a). A Reliable Assessment of 8-Oxo-3Deoxyguanosine Levels in Nuclear and Mitochondrial DNA using the Sodium Iodide Method to Isolate DNA.  Nucleic Acids Res. 29, 2117-2126.


Hamilton, M.L., Van Remmen, H., Drake, J.A., Yang, H., Guo, Z.M., Kewitt, K., Walter, C.A., and Richardson, A.  (2001b). Does Oxidative Damage to DNA Increase with Age?  Proc. Natl. Acad. Sci., USA. 98, 10469-10474.

We require snap frozen tissue stored at -80ºC for the oxo8dG assay. It is best not to store the tissue for more than a few months before conducting the assay to minimize potential oxidation of DNA during storage. For most tissues, 100-200 mg of tissue is optimal for analysis. However, for skeletal muscle ~500 mg of tissue is required (Hamilton et al. 2001).

8-oxo-deoxy guanosine: $100/sample (not currently available)

​Protein carbonyls

Protein Oxidation. Oxidative modifications to proteins can lead to mis-folded proteins that are prone to forming deleterious oligomers or aggregates that can alter cellular homeostasis and contribute to age-related pathologies and to the aging process itself. A predominant form of protein oxidative modification is the formation of carbonyl groups on specific amino acid residues, e.g., lysine, arginine, proline, and threonine. Carbonyl groups can also be formed by the reaction of amino acid residues with aldehydes (malondialdehyde, 4-hydroxy-2-nonenal) and reactive carbonyl derivatives generated through the reaction of reducing sugars or their oxidation products with lysine residues of proteins. The Core uses a modified assay in which oxidized proteins are measured using a fluorescent-based assay to detect protein carbonyls (Chaudhuri et al. 2006). This method is very sensitive and allows quantitative detection of proteins with even very low levels of protein carbonyls. Global changes in oxidized proteins are measured in tissue homogenates treated with fluorescein-5-thiosemicarbazide (FTC) and subjected to electrophoresis on a 12% gel to resolve fluorescence-labeled proteins from FTC. Importantly, samples can also be analyzed by two dimensional gel electrophoresis to identify carbonyl levels of individual proteins. The detailed methods for this analysis can be found in Chaudhuri et al, 2006 and Pierce et al., 2006.


References:


Chaudhuri, A.R., de Waal, E.M., Pierce, A., Van Remmen, H., Ward, W.F., and Richardson, A. (2006). Detection of Protein Carbonyls in Aging Liver Tissue: A Fluorescence-based Proteomic Approach. Mech. Age. Dev., 127, 894-861.


Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C.(1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

The tissue sample is homogenized in deaerated buffer [20 mM sodium phosphate buffer pH 6.0 containing 0.5 mM MgCl2, 1 mM EDTA and protease cocktail inhibitors (500 µM AEBSF, HCl, 150 nM aprotinin, 0.5 mM EDTA, disodium salt and 1 µM leupeptin hemisulfate)] and centrifuged at 4ºC for 1 hr at 100,000g. The resulting supernatant (cytosolic fraction) is treated with 1% streptomycin sulfate and incubated at 37ºC for 10 min. The solution is centrifuged at 11,000g for 10 min at room temperature to remove nucleic acids which contain reactive carbonyl groups. The protein concentration in the supernatant was measured by the Bradford assay, and used to measure protein carbonyl groups either before or after exposing the cytosolic extracts to an oxidative stress.


The cytosolic extracts are diluted to 1 mg/ml, and the extracts are then mixed with FTC (1 mM) and incubated at 37ºC for 150 min in the dark. The proteins are precipitated with an equal volume of 20% chilled TCA (v/v) and centrifuged at 16,000g for 5 min at 25ºC. The pellets are then re-suspended and washed five times with 100% ethanol/ethyl acetate (1:1) to remove the unbound FTC. The final pellets were then dissolved in phosphate buffer pH 8.0 containing 0.5 mM MgCl2, 1 mM EDTA and 8M urea. The concentration of the protein in each sample is measured by Bradford assay, and approximately 15-25 µg of protein is subjected to 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After electrophoresis, the image of the fluorescent protein on the gel is captured with the Typhoon 9400 using an excitation wavelength of 488 nm and an emission filter at 520 nm with a 40nm bandpass. The intensity of fluorescence for each lane (from top to bottom of the lane) was calculated using ImageQuant 5.0 (Molecular Dynamics, Amersham) software.


Carbonyl groups in specific proteins are determined using 2D gel electrophoresis as described by Chaudhuri et al., (2006).

We require snap frozen tissue stored at -80ºC for the protein carbonyl assay.  It is best not to store the tissue for more than a few months before conducting the assay to minimize potential oxidation of DNA during storage. For most tissues, 50 mg of tissue is optimal for analysis but depending on the tissue or sample type,  20 mg is often sufficient.

Protein carbonyls (specific proteins): $275/sample

Protein carbonyls (total): $80/sample

​Mitochondrial Function in cells & tissue

The Core provides investigators with services to measure mitochondrial function (oxidant generation, ATP production, respiration) as well as energy charge (ATP, ADP, and AMP) in fresh tissue samples or isolated mitochondria. This includes in vitro analyses of mitochondrial function in isolated mitochondria, respirometry analysis in fresh tissue using the Oroborus respirometer, as well as measurement of mitochondrial function in cells using the Seahorse XF24 Extracellular Flux Analyzer. While energy status can be measured on flash frozen tissues/cells, assays of mitochondria function must be performed with fresh tissue/cells or freshly-isolated mitochondria.

Additional assays offered as needed (contact Core Leader).


ETC activities. A limited number of users may be interested in measuring electron transport chain activities. Alterations in activity of the electron transport complexes can be measured using BN-PAGE (Schagger, 2001) or spectrophotometrically as we have previously described (Pulliam et al., 2014).


EPR Analysis. Rates of O2. and H2O2 production will be measured using EPR spectroscopy in permeabilized and intact mitochondria. For these studies, we will use a Bruker ESP300 spectrometer operating at 9.45 GHz with 10G field modulation at 100 kHz. The EPR is also equipped with liquid helium cooling, providing capability to measure the oxidation state and composition of protein iron sulfur centers such as exist in aconitase. For details see results from Dr. Van Remmen’s laboratory (Mansouri et al., 2006).

Assays of Mitochondrial Function. We offer investigators the ability to directly measure mitochondrial function in isolated mitochondria, tissue samples and in cultured cells. Alterations in mitochondrial function and increased mitochondrial ROS generation have long been implicated in the reduced capacity in cellular function that occurs with aging. We have optimized an array of assays to measure aspects of mitochondrial function, including ROS generation, mitochondrial respiration, and ATP production. Assays for measuring H2O2 and superoxide anion release, mitochondrial respiration and ATP production from isolated mitochondria are well established. ATP production and H2O2 release are measured in isolated mitochondria using a luciferase/luciferin based system and the fluorogenic probe, Amplex Red (Molecular Probes) respectively. Mitochondrial respiratory function is assessed in isolated mitochondria and permeabilized tissue using the Oroborus O2K high resolution respirometer which allows analysis of mitochondrial respiration without isolation of mitochondria. In addition to studies in isolated mitochondria, we also have the capability to measure oxygen consumption rate (OCR) in cultured cells using the Seahorse Extracellular Flux Analyzer. Many laboratories have used this methodology tomeasure mitochondrial function in cells. The Seahorse Analyzer also contains a probe to measure acidification of the media and to measure the proton production rate (PPR) or rate of extracellular acidification due to lactic acid production during glycolytic energy metabolism. By measuring OCR and PPR simultaneously, we can get a more detailed picture of cellular energetic flux through these two pathways. This technique is a non-invasive and high throughput analysis that allows us to measure the bioenergetic state and physiology of the cell without disruption of the cells. Energy Status. AMP, ADP, and ATP: NADH provides reducing equivalents for electron transport and, as such, levels of NADH and NAD+ are closely linked to mitochondrial function and energy homeostasis. We will measure AMP, ADP, and ATP concentrations to evaluate redox-associated changes in energy charge. Samples are extracted with 125 mM KOH and resolved by ion pairing reverse phase HPLC and quantified using UV/VIS detection.

Mitochondrial function requires fresh tissue or cells and therefore sample preparation will be discussed on a case by case basis with the Core Leader.

Mitochondrial Function Assays: Contact Core Leader for the cost of service

​In vivo free radical imaging

The in vivo and in situ immuno-spin trapping (IST)–molecular magnetic resonance imaging (mMRI) approach initially stemmed from combining the in vitro/ex vivo IST technique developed by Mason with in vivo imaging so that heterogeneous and tissue-specific levels of free radicals could be visualized in experimental animal disease models. To visualize free radicals in biological systems (e.g., cells and tissues), Mason et al. developed an anti-5,5-dimethyl-pyrroline-N-oxide (DMPO) antibody that could trap radicals, and then be visualized by a fluorescence tag. To visualize the DMPO-trapped radicals in vivo, our group decided to conjugate the anti-DMPO antibody to an MRI contrast agent construct so that DMPO-trapped radicals could be observed spatially either in two or three dimensions with a preclinical MRI system (10, 54–58). High-field MRI was considered due to the superior image resolution (e.g., ∼50 × 50 μm2 for mice and ∼100 × 100 μm2 for rats) of these imaging systems.

The approach involves the use of a spin-trapping agent, DMPO, which is used to trap free radicals in a disease model, and administration of an mMRI probe, called an anti-DMPO probe (Fig. 1), which combines an antibody against DMPO-radical adducts and an MRI contrast agent, resulting in targeted free radical adduct mMRI (see Fig. 2 for methodology scheme). The contrast agent used in the Towner approach includes an albumin-gadolinium diethylene tri-amine penta-acetic acid (Gd-DTPA)-biotin construct, where the anti-DMPO antibody is covalently linked to the cysteine residues of albumin, forming an anti-DMPO adduct antibody-albumin-Gd-DTPA-biotin entity. The Gd-DTPA moiety acts as the MRI signaling component, which will increase MRI signal intensity (SI) in a T1-weighted morphological MRI sequence, and decrease T1 relaxation in a T1 map image. Both of these parameters, MRI SI or T1 relaxation, can be used to assess the presence of the anti-DMPO probe. The biotin moiety can be used for ex vivo validation of the presence of the anti-DMPO probe in tissues, by using streptavidin fluorescence (e.g., Cy3) or streptavidin-horse radish peroxidase (HRP) to tag the biotin in the anti-DMPO probe.

Figure 1: Illustration of the anti-DMPO probe. Modified from Gomez-Mejiba et al. (17). anti-DMPO probe, anti-DMPO-adduct antibodyalbumin-Gd-DTPA-biotin; DMPO, 5,5-dimethylpyrroline-N-oxide.

Figure 2: Combined IST and free radical-targeted mMRI approach. Initially mice are administered DMPO (i.p.) to trap free radicals. Any cell membrane-bound radicals (e.g., oxidized proteins or lipids) can then be detected with the anti-DMPO probe (administered via a tail-vein catheter). Modified from Towner et al. (57). i.p., intraperitoneal; mMRI, molecular magnetic resonance imaging.


References


Mason RP. Assay of in situ radicals by electron spin resonance. Methods Enzymol. 105: 416422, 1984.


Mason RP. In vivo spin trapping – from chemistry to toxicology. In: Toxicology of the Human Environment: The Critical Role of Free Radicals. Edited by Rhodes C. London: Taylor and Francis. 2000, pp. 49-70.


Mason RP. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping. Free Radic Biol Med. 36: 1214-1223, 2004.


Mason RP. Imaging free radicals in organelles, cells, tissue, and in vivo with immuno-spin trapping. Redox Biol. 8: 422-9, 2016.


Gomez-Mejiba SE, Zhai Z, Akram H, Deterding LJ, Hensley K, Smith N, Towner RA, Tomer KB, Mason RP, Ramirez DC. Immuno-spin trapping of protein and DNA radicals: “tagging” free radicals to locate and understand the redox process. Free Radic Biol Med. 46: 853-65, 2009.


Gomez-Mejiba SE, Zhai Z, Della-Vedova MC, Muñoz MD, Chatterjee S, Towner RA, Hensley K, Floyd RA, Mason RP, Ramirez DC. Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals. Biochim Biophys Acta. 1840(2):722-9, 2014.


Khoo NK, Cantu-Medellin N, St Croix C, Kelley EE. In vivo immuno-spin trapping: imaging the footprints of oxidative stress. Curr Protoc Cytom. 74: 12.42.1-11, 2015.


Ramirez DC, Mason RP. Immuno-spin trapping: detection of protein-centered radicals. Curr Protoc Toxicol. 17: 17, 2005.


Coutinho de Souza P, Smith N, Atolagbe O, Ziegler J, Nijoku C, Lerner M, Ehrenshaft M, Mason RP, Meek B, Plafker SM, Saunders D, Mamedova N, Towner RA. OKN-007 decreases free radicals levels in a preclinical F98 rat glioma model. Free Radical Biol Med. 87: 157-168, 2015.


Towner RA, Smith N, Saunders D, Henderson M, Downum K, Lupu F, Silasi-Mansat R, Ramirez DC, Gomez-Mejiba SE, Bonini MG, Ehrenshaft M, Mason RP. In vivo imaging of immuno-spin trapped radicals with molecular MRI in a mouse diabetes model. Diabetes 61: 2405-13, 2012.


Towner RA, Smith N, Saunders D, Carrizales J, Lupu F, Silasi-Mansat R, Ehrenshaft M, Mason RP. In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice. Free Radic Res. 49: 1140-46, 2015.


Towner RA, Smith N, Saunders D, Lupu F, Silasi-Mansat R, West M, Ramirez DC, Gomez-Mejiba SE, Bonini MG, Mason RP, Ehrenshaft M, Hensley K. In vivo detection of free radicals using molecular MRI and immuno-spin-trapping in a mouse model for amyotrophic lateral sclerosis (ALS). Free Radic Biol Med. 63: 351-360, 2013.


Towner RA, Smith N, Saunders D, De Souza PC, Henry L, Lupu F, Silasi-Mansat R, Ehrenshaft M, Mason RP, Gomez-Mejiba SE, Ramirez DC. Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas. Biochim Biophys Acta. 1832: 2153-2161, 2013.


Towner RA, Garteiser P, Bozza F, Smith N, Saunders D, d’Avila JCP, Magno F, Oliveira MF, Ehrenshaft M, Lupu F, Silasi-Mansat R, Ramirez DC, Gomez-Mejiba SE, Mason RP, Faria-Neto HCC. In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin-trapping. Free Radica Biol Med. 65: 828-837, 2013.


Towner RA, Smith N. In vivo and in situ detection of macromolecular free radicals using immuno-spin trapping and molecular MRI. Antioxidants & Redox Signaling 2018; 28: 1404-1415.


Ahn B, Smith N, Saunders D, Ranjit R, Kneis P, Towner RA, Van Remmen H. Using MRI to measure in vivo free radical production and perfusion dynamics in a mouse model of elevated oxidative stress and neurogenic atrophy. Redox Biol. 2019 Sep;26:101308.

For in vivo analysis, if animals (e.g. mice or rats) are shipped to us, we can treat with DMPO and subsequently with the free radical-targeting molecular MRI probe (anti-DMPO probe) to detect macromolecular free radicals with our 7.0 T preclinical MRI system, or we can provide the anti-DMPO probe which can be used on your preferred preclinical MRI system, or alternately used in an oxidative stress model of your choice and then tissues/organs of interest are either fixed in formalin or stored at 4º C in PBS and shipped to our facility for ex vivo free radical detection. For fresh tissue samples, it is best not to store the tissue for more than a week before conducting the assay to minimize potential tissue damage during storage. Whole organ/tissue (if possible) is optimal for analysis.

Anti-DMPO probe: $80/mouse or $800/rat

In vivo/ex vivo free radicals: $275/sample

Service Request

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