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Free Radical and ROS Core Facility

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Free Radicals and Reactive Oxygen Species (ROS)

Free radicals are molecules possessing unpaired electrons and thus are reactive and short-lived in a biological setting. Dependent upon the site of and rate of production (concentration), free radicals can mediate both detrimental modifications to biomolecules and/or participate in homeostatic, cellular signal transduction.

Reactive oxygen species (ROS) comprise both free radical and non-free radical oxygen intermediates such as hydrogen peroxide (H2O2), superoxide (O2•-), singlet oxygen (1O2), and the hydroxyl radical (OH). These species are formed by the metabolism of oxygen in mitochondria, ionizing radiation, UV radiation, xenobiotic catabolism and a variety of cellular enzymes including Xanthine Oxidoreductase (XOR), NADPH oxidases (Nox), Aldehyde Oxidase (AO), mitochondrial electron transport proteins and dysfunctional (uncoupled) Nitric Oxide Synthases (NOS).  Once thought to play an exclusively negative role by inducing cellular/tissue damage, degrading bioactive nitric oxide (NO) and enhancing phagocytic antimicrobial action, ROS have recently been identified as critical mediators of cell signaling. For example, ROS participate in the signaling events controlling cell proliferation, hypertrophy, migration, wound healing, angiogenesis, neuronal signaling, vasomotor tone, and thyroid hormone metabolism. Therefore, there is broad interest in reliable and sensitive methods to detect and quantify these reactive species.

The Free Radical and ROS Facility is designed to aid investigators in the detection, quantification and imaging of reactive species in multiple model systems, as such we offer assistance with experimental design and execution of assays. We perform quantitative measurement of superoxide anion (O2•-), hydrogen peroxide (H2O2) and analyses of biomarkers of oxidative stress by imaging.

Samples can be live cells and tissue –or- homogenates of fresh-frozen cells and tissues.



Detection and quantification of ROS in whole cells in culture as well as in cell and tissue homogenates using spectrophotometric, chemiluminescence and fluorescence techniques on a 96- or 384-well platform


SOD-inhibitable cytochrome C assay (cell and tissue homogenates)

  • Classic method for detection of O2.-
  • Detection of extracellular O2.-
  • Not highly sensitive
  • Suitable for cell-free assays

Hydropropidine (HPr+) assay (live cells, cell and tissue homogenates)

  • Cell-impermeant analog of hydroethidine
  • Detection of extracellular O2.-


Amplex Red assay (live cells, cell and tissue homogenates)

  • Extracellular detection of  H2O2
  • Fluorescence assay [540nm (excitation) and 580 nm (emission)]

Coumarin boronate acid (CBA) assay (live cells, cell and tissue homogenates)

  • End-point
  • Specific for H2O2
  • Fluorescence assay [315nm (excitation) and 425 nm (emission)]

ASSAYS FOR ONOO- (peroxynitrite)

  • CBA Assay


  • Chemiluminescence assay
  • Detection of superoxide anion and peroxynitrite anion
  • Highly sensitive
  • Ideal for early-stage, high throughput assays that are corroborated with more trusted and validated assays listed above.

*All results are confirmed using more than one assay.


Detection of 4-Hydroxynonenal (4-HNE)

4-HNE is one of the major end byproducts of lipid peroxidation and is considered a biomarker of oxidative stress

Detection of 3-nitrotyrosine (3-NT)

Nitrotyrosine is a product of tyrosine nitration mediated by reactive nitrogen species including peroxynitrite anion (also considered a good marker for oxidative stress).




Measurements of protein and mRNA expression for various enzymatic sources of ROS, i.e. NADPH oxidase (Nox) core components and their multiple additional subunits, xanthine oxidase, etc.


Implementation, development and validation of other methods are in progress, these include:


    • DHE-HPLC: for detection of O2•-.
    • Imaging of DMPO adducts.
    • Fluorescent protein-based redox sensitive probes including HyPer, roGFPs for detection of subcellular ROS (mitochondrial, cytosolic).
    • We also have the capability and expertise to develop and optimize other plate-based biochemical assays using spectrophotometric absorbance, fluorescence and luminescence detection to suit the needs of the project.


Eugenia Cifuentes-Pagano, PhD

Eugenia Cifuentes-Pagano, PhD

Research Assistant Professor
Department of Pharmacology and Chemical Biology

412-648-2610 |
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Publications utilizing the VMI Free Radical and ROS Core Facility

Li Y*, Cifuentes-Pagano E*, DeVallance ER, de Jesus DS, Sahoo S,  Meijles DN, Ross M, Koes D, Camacho  C, St  Croix  C., Pagano  PJ. (2019) NADPH oxidase 2 inhibitors CPP11G and CPP11H attenuate endothelial cell inflammation & vessel dysfunction and restore mouse hind-limb flow. Redox Biol. 2019 Feb 15; 22:101143. doi: 10.1016/j.redox.2019.101143. [Epub ahead of print] PMID: 30897521

de Jesus DS, DeVallance E, Li Y, Falabella M, Guimaraes D, Shiva S, Kaufman BA, Gladwin MT, Pagano PJ. (2019) Nox1/Ref-1-mediated activation of CREB promotes Gremlin1-driven endothelial cell proliferation and migration. Redox Biol. 22:101138. doi: 10.1016/j.redox.2019.101138. [Epub ahead of print] PMID: 30802716

Vara D, Cifuentes-Pagano E, Pagano PJ, Pula G. (2019) A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli. Haematologica. pii: haematol.2018.208819. doi: 10.3324/haematol.2018.208819. PMID: 30679320

Robinson AR, Yousefzadeh MJ, Rozgaja TA, Wang J, Li X, Tilstra JS, Feldman CH, Gregg SQ, Johnson CH, Skoda EM, Frantz MC, Bell-Temin H, Pope-Varsalona H, Gurkar AU, Nasto LA, Robinson RAS, Fuhrmann-Stroissnigg H, Czerwinska J, McGowan SJ, Cantu-Medellin N, Harris JB, Maniar S, Ross MA, Trussoni CE, LaRusso NF, Cifuentes-Pagano E, Pagano PJ, Tudek B, Vo NV, Rigatti LH, Opresko PL, Stolz DB, Watkins SC, Burd CE, Croix CMS, Siuzdak G, Yates NA, Robbins PD, Wang Y, Wipf P, Kelley EE, Niedernhofer LJ. (2018) Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging. Redox Biol. 17:259-273. doi: 10.1016/j.redox.2018.04.007. Epub 2018 Apr 13. PMID: 29747066

Meijles DN, Sahoo S, Al Ghouleh I, Amaral JH, Bienes-Martinez R, Knupp HE, Attaran S, Sembrat JC, Nouraie SM, Rojas MM, Novelli EM, Gladwin MT, Isenberg JS, Cifuentes-Pagano E, Pagano PJ. (2017) The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1. Sci Signal. 10(501). pii: eaaj1784. doi: 10.1126/scisignal.aaj1784. PMID: 29042481

Ghouleh IA, Sahoo S, Meijles DN, Amaral JH, de Jesus DS, Sembrat J, Rojas M, Goncharov DA, Goncharova EA, Pagano PJ. (2017) Endothelial Nox1 oxidase assembly in human pulmonary arterial hypertension; driver of Gremlin1-mediated proliferation. Clin Sci (Lond). 131(15):2019-2035. doi: 10.1042/CS20160812. Print 2017 Aug 1. PMID: 28522681

Csányi G, Feck DM, Ghoshal P, Singla B, Lin H, Nagarajan S, Meijles DN, Al Ghouleh I, Cantu-Medellin N, Kelley EE, Mateuszuk L, Isenberg JS, Watkins S, Pagano PJ. (2017) CD47 and Nox1 Mediate Dynamic Fluid-Phase Macropinocytosis of Native LDL. Antioxid Redox Signal. 26(16):886-901. doi: 10.1089/ars.2016.6834. Epub 2017 Jan 31. PMID: 27958762

Al Ghouleh I, Meijles DN, Mutchler S, Zhang Q, Sahoo S, Gorelova A, Henrich Amaral J, Rodríguez AI, Mamonova T, Song GJ, Bisello A, Friedman PA, Cifuentes-Pagano ME, Pagano PJ. (2016) Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype. Proc Natl Acad Sci U S A. 113(36):E5308-17. doi: 10.1073/pnas.1514161113. Epub 2016 Aug 18. PMID: 27540115

Sahoo S, Meijles DN, Al Ghouleh I, Tandon M, Cifuentes-Pagano E, Sembrat J, Rojas M, Goncharova E, Pagano PJ. (2016) MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension. PLoS One.11(5):e0153780. doi: 10.1371/journal.pone.0153780. eCollection 2016. PMID: 27144530

Vogel S, Bodenstein R, Chen Q, Feil S, Feil R, Rheinlaender J, Schäffer TE, Bohn E, Frick JS, Borst O, Münzer P, Walker B, Markel J, Csanyi G, Pagano PJ, Loughran P, Jessup ME, Watkins SC, Bullock GC, Sperry JL, Zuckerbraun BS, Billiar TR, Lotze MT, Gawaz M, Neal MD. (2015) Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest. 125(12):4638-54. doi: 10.1172/JCI81660. Epub 2015 Nov 9. PMID: 26551681

Quesada IM, Lucero A, Amaya C, Meijles DN, Cifuentes ME, Pagano PJ, Castro C. (2015). Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques. Atherosclerosis. 242(2):469-75. doi: 10.1016/j.atherosclerosis.2015.08.011. Epub 2015 Aug 8. PMID: 26298737

Mukawera E, Chartier S, Williams V, Pagano PJ, Lapointe R, Grandvaux N. (2015) Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines. Redox Biol. 6:9-18. doi: 10.1016/j.redox.2015.06.010. Epub 2015 Jun 23. PMID: 26177467

Rodríguez AI, Csányi G, Ranayhossaini DJ, Feck DM, Blose KJ, Assatourian L, Vorp DA, Pagano PJ. (2015) MEF2B-Nox1 signaling is critical for stretch-induced phenotypic modulation of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 35(2):430-8. doi: 10.1161/ATVBAHA.114.304936. Epub 2014 Dec 30.
PMID: 25550204

Hahn NE, Musters RJ, Fritz JM, Pagano PJ, Vonk AB, Paulus WJ, van Rossum AC, Meischl C, Niessen HW, Krijnen PA. (2014) Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells. Cell Signal. 26(9):1818-24. doi: 10.1016/j.cellsig.2014.04.018. Epub 2014 May 2. PMID: 24794531

Yao M, Rogers NM, Csányi G, Rodriguez AI, Ross MA, St Croix C, Knupp H, Novelli EM, Thomson AW, Pagano PJ, Isenberg JS. (2014) Thrombospondin-1 activation of signal-regulatory protein-α stimulates reactive oxygen species production and promotes renal ischemia reperfusion injury. J Am Soc Nephrol. 25(6):1171-86. doi: 10.1681/ASN.2013040433. Epub 2014 Feb 7. Erratum in: J Am Soc Nephrol. 2014 Aug;25(8):1884. PMID: 24511121

Cifuentes-Pagano E, Saha J, Csányi G, Ghouleh IA, Sahoo S, Rodríguez A, Wipf P, Pagano PJ, Skoda EM. (2013) Bridged tetrahydroisoquinolines as selective NADPH oxidase 2 (Nox2) inhibitors. Med Chem Comm. 4(7):1085-1092. PMID: 24466406

Frazziano G, Al Ghouleh I, Baust J, Shiva S, Champion HC, Pagano PJ. (2014) Nox-derived ROS are acutely activated in pressure overload pulmonary hypertension: indications for a seminal role for mitochondrial Nox4. Am J Physiol Heart Circ Physiol. 306(2):H197-205. doi: 10.1152/ajpheart.00977.2012. Epub 2013 Nov 8. PMID:24213612

Ranayhossaini DJ, Rodriguez AI, Sahoo S, Chen BB, Mallampalli RK, Kelley EE, Csanyi G, Gladwin MT, Romero G, Pagano PJ. (2013) Selective recapitulation of conserved and non-conserved regions of putative NOXA1 protein activation domain confers isoform-specific inhibition of Nox1 oxidase and attenuation of endothelial cell migration. J Biol Chem. 288(51):36437-50. doi: 10.1074/jbc.M113.521344. Epub 2013 Nov 1. PMID:24187133

Al Ghouleh I, Rodríguez A, Pagano PJ, Csányi G. (2013)  Proteomic analysis identifies an NADPH oxidase 1 (Nox1)-mediated role for actin-related protein 2/3 complex subunit 2 (ARPC2) in promoting smooth muscle cell migration.  Int J Mol Sci. 14(10):20220-35. doi: 10.3390/ijms141020220.  PMID:24152438.

Csányi G, Pagano PJ. (2013)  Strategies Aimed at Nox4 Oxidase Inhibition Employing Peptides from Nox4 B-Loop and C-Terminus and p22 (phox) N-Terminus: An Elusive Target. Int J Hypertens. 2013:842827. doi: 10.1155/2013/842827. Epub 2013 Mar 31.  PMID: 23606947

Csányi G, Yao M, Rodríguez AI, Al Ghouleh I, Sharifi-Sanjani M, Frazziano G, Huang X, Kelley EE, Isenberg JS, Pagano PJ. Thrombospondin-1 regulates blood flow via CD47 receptor-mediated activation of NADPH oxidase 1. Arterioscler Thromb Vasc Biol. 2012 Dec;32(12):2966-73. doi: 10.1161/ATVBAHA.112.300031. Epub 2012 Oct 18. PMID:23087362.

Al Ghouleh I, Frazziano G, Rodriguez AI, Csányi G, Maniar S, St Croix CM, Kelley EE, Egaña LA, Song GJ, Bisello A, Lee YJ, Pagano PJ. (2013) Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy. Cardiovasc Res. 97(1):134-42. doi: 10.1093/cvr/cvs295. Epub 2012 Sep 20. PMID:22997161.

Bernard ME, Kim H, Rwigema JC, Epperly MW, Kelley EE, Murdoch GH, Dixon T, Wang H, Greenberger JS. (2011). Role of the esophageal vagus neural pathway in ionizing radiation induced seizures in nitric oxide synthase-1 homologous recombinant negative (NOS1-/-) mice. In Vivo. 25(6):861-9. PMID: 22021678.

Hahm ER, Barbi de Moura M, Kelley EE, Shiva S, Van Houten B, Singh SV. (2011). Withaferin A-induced apoptosis in human breast cancer cells is mediated by ROS-dependent activation of Bak., PLoS ONE, 6(8):e23354. PMID: 21853114.

Al Ghouleh I, Khoo NK, Knaus UG, Griendling KK, Touyz RM, Thannickal VJ, Barchowsky A, Nauseef WM, Kelley EE, Bauer PM, Darley-Usmar V, Shiva S, Cifuentes-Pagano E, Freeman BA, Gladwin MT, Pagano PJ. (2011). Oxidases and Peroxidases in Cardiovascular and Lung Disease: New Concepts in Reactive Oxygen Species Signaling. Free Rad. Biol. Med., 51(7):1271-88. PMID: 21722728.

Donadee C, Raat NHJ, Tejero J, Lee JS, Kelley EE, Zhao X, Liu C, Reynolds H, Azarov I, Frizzell S, Meyer EM, Donnenberg AD, Qu L, Triulzi D, Kim-Shapiro DB, Gladwin MT. (2011). Nitric oxide scavenging by red cell microparticles and cell free hemoglobin as a mechanism for the red cell storage lesion. Circulation, 124(4):465-76, 2011.  PMID: 21747051.

Malik UZ, Hundley NJ, Radi R, Freeman BA, Tarpey MM, Kelley EE. (2011). Febuxostat inhibition of endothelial-bound XO: implications for targeting vascular ROS production. Free Rad. Biol. Med. 51(1):179-84, 2011.  PMID: 21554948.

Manni ML, Tomai LP, Norris CL, Thomas LM, Kelley EE, Salter RD, Crapo JD, Chang LY, Freeman BA, Watkins SC, Pignellie JD, Oury, TD. (2011). Extracellular Superoxide Dismutase Attenuates Bacterial Pneumonia by Promoting Phagocytosis. American Journal of Pathology. 178:2752-9.  PMID: 21641397.

Csányi G, Cifuentes-Pagano E, Egaña L, Al Ghouleh I, Lopes LR, Kelley EE, Pagano PJ. (2011). Nox2 B-loop peptide, Nox2ds, specifically inhibits Nox2 oxidase. Free Rad Biol. Med., 51(6):1116-25.  PMID: 21586323.

Alef W, Vallabhaneni R, Carchman E, Morris S, Shiva S, Wang Y, Kelley EE, Tarpey MM, Gladwin M, Tzeng E, Zuckerbraun B. (2011). Nitrite-Generated Nitric Oxide Circumvents Dysregulated Arginine/Nitric Oxide Synthase Signaling To Protect Against Intimal Hyperplasia. Journal of Clinical Investigation. 121(4):1646-56.  PMID: 21436585.

Xiao D, Powolny AA, Moura MB, Kelley EE, Bommareddy A, Kim SH, Hahm ER, Normolle D, Van Houten B, Singh SV. (2010). Phenethyl Isothiocyanate Inhibits Oxidative Phosphorylation to Trigger Reactive Oxygen Species-mediated Death of Human Prostate Cancer Cells. J Biol Chem. 285:26558-69. PMID: 20571029.

Kelley EE, Khoo NKH, Hundley NJ, Malik UZ, Freeman BA, Tarpey MM. (2010). Hydrogen peroxide is the major oxidant product of xanthine oxidase. Free Rad. Biol. Med. 48(4):493-8. PMID: 19941951.

Rodriguez AI, Gangopadhyay A, Kelley EE, Pagano PJ, Zuckerbraun BS, Bauer PM. (2009). HO-1 and CO Decrease Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cell Migration Via Inhibition of Nox1. Arterioscler Thromb Vasc Biol. 30(1):98-104. PMID:19875720.