RESEARCH PRODUCTS/Genetically-encoded fluorescent sensors:

    HyPer

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References citing HyPer:
  • Sartoretto JL, Kalwa H, Pluth MD, Lippard SJ, Michel T.
    Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis.
    Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):15792-7
    pmid: 21896719
     
  • Morinaka A, Yamada M, Itofusa R, Funato Y, Yoshimura Y, Nakamura F, Yoshimura T, Kaibuchi K, Goshima Y, Hoshino M, Kamiguchi H, Miki H.
    Thioredoxin mediates oxidation-dependent phosphorylation of CRMP2 and growth cone collapse.
    Sci Signal. 2011 Apr 26;4(170):ra26
    pmid: 21521879
     
  • Elsner M, Gehrmann W, Lenzen S.
    Peroxisome-generated hydrogen peroxide as important mediator of lipotoxicity in insulin-producing cells.
    Diabetes. 2011 Jan;60(1):200-8
    pmid: 20971967
     
  • Miller EW, Dickinson BC, Chang CJ.
    Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling.
    Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15681-6
    pmid: 20724658
  1. Constructs used: cytosolic HyPer (pHyPer-cyto vector, Evrogen).
  2. Expression system: HEK 293 cells and HeLa cells transiently transfected with plasmid using Lipofectamine 2000 or Lipofectamine LTX reagents.
  3. Detection system: increase in HyPer signal in response to H2O2 was detected with Axiovert 200M (equipped with an AxioCamMRm). Excitation was provided by an X-Cite series 120 lamp using a filter cube containing a bandpass excitation filter at 470/40nm, a beam splitter at 495nm, and a bandpass emission filter at 525/50nm.
  • Narayanan D, Xi Q, Pfeffer LM, Jaggar JH.
    Mitochondria control functional CaV1.2 expression in smooth muscle cells of cerebral arteries.
    Circ Res. 2010 Sep 3;107(5):631-41
    pmid: 20616314
  1. Constructs used: cytosolic HyPer (pHyPer-cyto vector, Evrogen).
  2. Expression system: myocytes of intact cerebral arteries transfected with plasmid using reverse permeabilization.
  3. Detection system: increase in HyPer signal in response to H2O2 was detected with Zeiss LSM5 laser-scanning confocal microscope. HyPer excited by 488nm laser and signal detected with 510nm LP emission filter.
  • Wu RF, Ma Z, Liu Z, Terada LS.
    Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local Ras activation.
    Mol Cell Biol. 2010 Jul;30(14):3553-68
    pmid: 20457808
  1. Constructs used: 1) cytosolic HyPer (pHyPer-cyto vector, Evrogen);
    2) mitochondria-targeted HyPer (pHyPer-dMito vector, Evrogen);
    3) ER-targeted HyPer (30a.a. signal sequence of bovine preprolactin and ER retention signal KDEL fused to the HyPer N- and C-terminus respectively).
  2. Expression system: human umbilical vein endothelial cells (HUVEC) transduced with lentiviral or adenoviral particles.
  3. Detection system: Nikon TE2000-U (TIRF microscope). HyPer was sequentially excited at 405nm (405/40 bandpass filter) and 492nm (492/18 bandpass filter). HyPer signal was detected with 530/35 bandpass emission filter. H2O2 levels were evaluated in individual cells as ratio of HyPer signal detected after excitation at 492nm and 405 nm respectively (492/405nm ratio).
  • Ameziane-El-Hassani R, Boufraqech M, Lagente-Chevallier O, Weyemi U, Talbot M, Métivier D, Courtin F, Bidart JM, El Mzibri M, Schlumberger M, Dupuy C.
    Role of H2O2 in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells.
    Cancer Res. 2010 May 15;70(10):4123-32
    pmid: 20424115
  1. Constructs used: 1) cytosolic HyPer (pHyPer-cyto vector, Evrogen);
    2) nuclear-targeted HyPer (pHyPer-nuc vector, Evrogen).
  2. Expression system: human thyroid epithelial cell line (HTori-3) transiently transfected with JetPEI reagent.
  3. Detection system: increase in HyPer signal in response to H2O2 was detected with Zeiss LSM510 confocal microscope. HyPer excitation by 488nm laser and emission detected at 530nm.
  • Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, Pozzan T, Lo Schiavo F.
    H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scavenging system.
    Plant J. 2010 Jun 1;62(5):760-72
    pmid: 20230493
  1. Constructs used: 1) for cytoplasmic localization: humanized HyPer coding sequence (from pHyPer-cyto vector, Evrogen) under the control of double 35S promoter and TEV translational enhancer;
    2) for peroxisomal localization: peroxisome localization signal (KSRM) fused to the HyPer C-terminus.
  2. Expression system: epidermal leaf cells from transgenic plants (Arabidopsis thaliana) generated with Agrobacterium.
  3. Detection system: Nikon PCM laser scanning confocal microscope. HyPer excitation at 488 nm and detection of emission at 530/560 nm.
  • Ungvari Z, Labinskyy N, Mukhopadhyay P, Pinto JT, Bagi Z, Ballabh P, Zhang C, Pacher P, Csiszar A.
    Resveratrol attenuates mitochondrial oxidative stress in coronary arterial endothelial cells.
    Am J Physiol Heart Circ Physiol. 2009 Nov;297(5):H1876-81
    pmid: 19749157
  1. Constructs used: pHyper-dMito vector, Evrogen.
  2. Expression system: primary human coronary arterial endothelial cells (CAECs) transfected by Amaxa Nucleofector technology.
  3. Detection system: Tecan Infinite M200 plate reader.
  • Jin BY, Sartoretto JL, Gladyshev VN, Michel T.
    Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.
    Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17343-8
    pmid: 19805165
  1. Constructs used: pHyPer-cyto vector, Evrogen.
  2. Expression system: transiently transfected bovine aortic endothelial cells (BAEC) using Lipofectamine 2000.
  3. Detection system: Nikon TE2000 microscope.
  • Niethammer P, Grabher C, Look AT, Mitchison TJ.
    A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
    Nature. 2009 Jun 18;459(7249):996-9
    pmid: 19494811
  1. Constructs used: mRNA encoding HyPer.
  2. Expression system: zebrafish embryos.
  3. Detection system: used for HyPer excitation 420/40 and 501/16 bandpass filters. HyPer emission acquired using 535/30 bandpass filter. Ratiometric measurement of H2O2 levels based on the HyPer500/HyPer420 ratio.
  • Treiber N, Peters T, Sindrilaru A, Huber R, Kohn M, Menke A, Briviba K, Kreppel F, Basu A, Maity P, Koller M, Iben S, Wlaschek M, Kochanek S, Scharffetter-Kochanek K.
    Overexpression of manganese superoxide dismutase in human dermal fibroblasts enhances the contraction of free floating collagen lattice: implications for ageing and hyperplastic scar formation.
    Arch Dermatol Res. 2009 Apr;301(4):273-87
    pmid: 19306099
  1. Constructs used: 1) for cytoplasmic localization: HyPer coding sequence (pHyPer-cyto vector, Evrogen) subcloned into adenoviral vector;
    2) for mitochondria localization: HyPer-dMito coding sequence (pHyPer-dMito vector, Evrogen) subcloned into adenoviral vector.
  2. Expression system: fibroblast cell lines transfected with adenoviral particles.
  3. Detection system: 24 hours after transfection evaluated H2O2 levels by FACS as a ratio of MFI from HyPer excited by 488nm and 405nm lasers.
  • Espinosa A, Garcia A, Hartel S, Hidalgo C, Jaimovich E.
    NADPH oxidase and hydrogen peroxide mediate insulin-induced calcium increase in skeletal muscle cells.
    J Biol Chem. 2009 Jan 23;284(4):2568-75
    pmid: 19028699
  1. Constructs used: pHyPer-cyto vector, Evrogen.
  2. Expression system: primary culture of mouse skeletal muscle cells.
  3. Detection system: ROS signalling; increase in HyPer fluorescence in response to insulin.
     
  • Markvicheva KN, Bogdanova EA, Staroverov DB, Lukyanov S, Belousov VV.
    Imaging of intracellular hydrogen peroxide production with HyPer upon stimulation of HeLa cells with EGF.
    Methods Mol Biol. 2009;476:76-83
    pmid: 19253046
  1. Constructs used: pHyPer-cyto vector, Evrogen.
  2. Expression system: HeLa cells transiently transfected with plasmid using FuGENE6 reagent.
  3. Detection system: Leica confocal system TCS SP2 (excitation for HyPer – laser 488nm, 5-15% power, emission detected 500-530 nm). H2O2 production (increase in green fluorescence from HyPer) was visualized in individual HeLa cells 24 hours after transfection in response to stimulation with EGF.
  • Myers D, Terada L.
    Visualization of Reactive Oxygen Species Production at Focal Adhesions.
    Am J Respir Crit Care Med. 2009; 179(1_MeetingAbstracts):A4166
     
  • Powers SK, Jackson MJ.
    Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production.
    Physiol Rev. 2008 Oct;88(4):1243-76.
    pmid: 18923182
     
  • Souslova EA, Chudakov DM.
    Genetically encoded intracellular sensors based on fluorescent proteins.
    Biochemistry (Mosc). 2007 Jul;72(7):683-97
    pmid: 17680759
     
  • Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S.
    Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.
    Nat Methods. 2006 Apr;3(4):281-6
    pmid: 16554833
  1. Constructs used: pQE-30-HyPer vector (for expression in E. coli) and pHyPer-cyto vector (for expression in mammalian cells).
  2. Expression system: E. coli; transiently transfected HeLa and PC12 cell lines; stably transfected COS7 cell line (selected with 1 mg/ml G418).
  3. Detection system: 1) Leica confocal system TCS SP2 (488-nm excitation laser, 4% power, emission detected in 500-520 nm range);
    2) FACS (excitation with 405-nm or 488-nm lasers and emission detected with 530/540nm filter).

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