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RESEARCH PRODUCTS/Genetically-encoded fluorescent sensors:

    HyPer

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References citing HyPer:
  • Corteselli EM, Samet JM, Gibbs-Flournoy EA.
    Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors.
    J Vis Exp. 2018 Feb 7;(132.) doi: 10.3791/56945
    pmid: 29443110
     
  • Bogdanova YA, Schultz C, Belousov VV.
    Local Generation and Imaging of Hydrogen Peroxide in Living Cells.
    Curr Protoc Chem Biol. 2017 Jun 19;9(2):117-127 doi: 10.1002/cpch.20
    pmid: 28628200
     
  • Huang K, Caplan J, Sweigard J, Czymmek K, Donofrio N.
    Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.
    Mol Plant Pathol. 2016 Mar 7.; doi: 10.1111/mpp.12392
    pmid: 26950262
     
  • Huang BK, Langford TF, Sikes HD.
    Using sensors and generators of H2O2 to elucidate the toxicity mechanism of piperlongumine and phenethyl isothiocyanate.
    Antioxid Redox Signal. 2016 Feb 23;[Epub ahead of print]
    pmid: 26905788
     
  • Cameron WD, Bui CV, Hutchinson A, Loppnau P, Gräslund S, Rocheleau JV.
    Apollo-NADP : a spectrally tunable family of genetically encoded sensors for NADP.
    Nat Methods. 2016 Feb 15;[Epub ahead of print] doi: 10.1038/nmeth.3764
    pmid: 26878383
     
  • He T, Hatem E, Vernis L, Lei M, Huang ME.
    PRX1 knockdown potentiates vitamin K3 toxicity in cancer cells: a potential new therapeutic perspective for an old drug.
    J Exp Clin Cancer Res. 2015 Dec 21;34(1):152 doi: 10.1186/s13046-015-0270-2
    pmid: 26689287
     
  • Warren EA, Netterfield TS, Sarkar S, Kemp ML, Payne CK.
    Spatially-resolved intracellular sensing of hydrogen peroxide in living cells.
    Sci Rep. 2015 Nov 20;5:16929 doi: 10.1038/srep16929
    pmid: 26585385
     
  • Forkink M, Basit F, Teixeira J, Swarts HG, Koopman WJ, Willems PH.
    Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells.
    Redox Biol. 2015 Oct 23;6:607-616 doi: 10.1016/j.redox.2015.09.003
    pmid: 26516986
     
  • Mishina NM, Mishin AS, Belyaev Y, Bogdanova EA, Lukyanov S, Schultz C, Belousov VV.
    Live-Cell STED Microscopy with Genetically Encoded Biosensor.
    Nano Lett. 2015 May 13;15(5):2928-32 doi: 10.1021/nl504710z
    pmid: 25871892
     
  • Lim JB, Sikes HD.
    Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity.
    Protein Eng Des Sel. 2015 Mar;28(3):79-83 doi: 10.1093/protein/gzv003
    pmid: 25691762
     
  • Hernández-Barrera A, Velarde-Buendía A, Zepeda I, Sanchez F, Quinto C, Sánchez-Lopez R, Cheung AY, Wu HM, Cardenas L.
    Hyper, a hydrogen peroxide sensor, indicates the sensitivity of the Arabidopsis root elongation zone to aluminum treatment.
    Sensors (Basel). 2015 Jan 6;15(1):855-67 doi: 10.3390/s150100855
    pmid: 25569758
     
  • Gehrmann W, Würdemann W, Plötz T, Jörns A, Lenzen S, Elsner M.
    Antagonism Between Saturated and Unsaturated Fatty Acids in ROS Mediated Lipotoxicity in Rat Insulin-Producing Cells.
    Cell Physiol Biochem. 2015;36(3):852-65 doi: 10.1159/000430261
    pmid: 26044490
     
  • Huang BK, Sikes HD.
    Quantifying intracellular hydrogen peroxide perturbations in terms of concentration.
    Redox Biol. 2014 Aug 6;2C:955-962 doi: 10.1016/j.redox.2014.08.001
    pmid: 25460730
     
  • Cheng WY, Larson JM, Samet JM.
    Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy.
    Methods. 2014 Mar 15;66(2):345-52 doi: 10.1016/j.ymeth.2013.06.008
    pmid: 23816786
  1. Redox imaging using both HyPer and roGFP simultaneously with subsequent signals separation.
  2. Constructs used: pHyPer-cyto, pHyPer-dMito.
  3. Expression system: BEAS-2B human airway epithelial cells.
  4. Detection system: Nikon Eclipse C1si confocal imaging system installed on an Eclipse Ti microscope, Nikon 60x Plan Apo VC 1.4 objective. Fluorescence excited with 10 mW 488 nm and a 17 mW 404 nm lasers with the emission spectra monitored from 490 nm to 570 nm over 32 channels.
  • Matlashov MV, Belousov VV, Enikolopov G.
    How Much H2O2 Is Produced by Recombinant D-Amino Acid Oxidase in Mammalian Cells?
    Antioxid Redox Signal. 2014 Mar 1;20(7):1039-44 doi: 10.1089/ars.2013.5618
    pmid: 24020354
  1. D-Alanine oxidase is used for controlled hydrogen peroxide production.
  2. Constructs used: pHyPer-cyto.
  3. Expression system: Hela-Kyoto and NIH-3T3 cells.
  4. Detection system: Leica 6000 widefield microscope equipped with a 20x air objective and HCX PL APO lbd.BL 63 x 1.4NA oil objective. Fluorescence was excited sequentially using 427/10 and 504/12 band-pass excitation filters, emission was collected using a 525/50 bandpass emission filter.
  • Lukyanov KA, Belousov VV.
    Genetically encoded fluorescent redox sensors.
    Biochim Biophys Acta. 2014 Feb;1840(2):745-56 doi: 10.1016/j.bbagen.2013.05.030
    pmid: 23726987
  1. Review
  • Kaludercic N, Carpi A, Nagayama T, Sivakumaran V, Zhu G, Lai EW, Bedja D, De Mario A, Chen K, Gabrielson KL, Lindsey ML, Pacak K, Takimoto E, Shih JC, Kass DA, Di Lisa F, Paolocci N.
    Monoamine Oxidase B Prompts Mitochondrial and Cardiac Dysfunction in Pressure Overloaded Hearts.
    Antioxid Redox Signal. 2014 Jan 10;20(2):267-80 doi: 10.1089/ars.2012.4616
    pmid: 23581564
  1. HyPer was used to localize H2O2 production in cells.
  2. Constructs used: pHyPer-cyto and pHyPer-dMito.
  3. Expression system: Neonatal rat cardiomyocytes.
  4. Detection system: Zeiss LSM 510 Meta.
  • Kruzel ML, Actor JK, Zimecki M, Wise J, Płoszaj P, Mirza S, Kruzel M, Hwang SA, Ba X, Boldogh I.
    Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression.
    J Biotechnol. 2013 Dec;168(4):666-75 doi: 10.1016/j.jbiotec.2013.09.011
    pmid: 24070904
  1. Recombinant lactoferrin protects from oxidative stress, induced by externally added glucose oxidase.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: Chinese hamster ovary cells.
  4. Detection system: FLx800 microplate reader.
  • Bertolotti M, Bestetti S, García-Manteiga JM, Medraño-Fernandez I, Dal Mas A, Malosio ML, Sitia R.
    Tyrosine kinase signal modulation: a matter of H2O2 membrane permeability?
    Antioxid Redox Signal. 2013 Nov 1;19(13):1447-51 doi: 10.1089/ars.2013.5330
    pmid: 23541115
     
  • Koczurkiewicz P, Podolak I, Skrzeczyńska-Moncznik J, Sarna M, Wójcik KA, Ryszawy D, Galanty A, Lasota S, Madeja Z, Czyż J, Michalik M.
    Triterpene saponosides from Lysimachia ciliata differentially attenuate invasive potential of prostate cancer cells.
    Chem Biol Interact. 2013 Oct 25;206(1):6-17 doi: 10.1016/j.cbi.2013.08.003
    pmid: 23954719
  1. ROS production in cancer cells in response to apoptotic stimuli.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: DU-145 cells.
  4. Detection system: Leica DMI6000B microscope, equipped with Hamamatsu EM-CCD camera, dual bandpass filter (D415/30x and HQ485/15x) for excitation and HQ535/48m filter for emission.
  • Ronen M, Shalaby S, Horwitz BA.
    Role of the transcription factor ChAP1 in cytoplasmic redox homeostasis: imaging with a genetically encoded sensor in the maize pathogen Cochliobolus heterostrophus.
    Mol Plant Pathol. 2013 Oct;14(8):786-90 doi: 10.1111/mpp.12047
    pmid: 23745603
  1. Constructs used: HyPer was expressed under the control of a fungal promoter, PTrpC, from pUCATPH.
  2. Expression system: Cochliobolus heterostrophus.
  3. Detection system: Zeiss LSM 700 confocal microscope.
  • Enyedi B, Zana M, Donkó Á, Geiszt M.
    Spatial and temporal analysis of NADPH oxidase-generated hydrogen peroxide signals by novel fluorescent reporter proteins.
    Antioxid Redox Signal. 2013 Aug 20;19(6):523-34 doi: 10.1089/ars.2012.4594
    pmid: 23121369
     
  • Grisham MB.
    Methods to detect hydrogen peroxide in living cells: Possibilities and pitfalls.
    Comp Biochem Physiol A Mol Integr Physiol. 2013 Aug;165(4):429-38 doi: 10.1016/j.cbpa.2013.02.003
    pmid: 23396306
  1. Review
  • Espinosa A, Campos C, Díaz-Vegas A, Galgani JE, Juretic N, Osorio-Fuentealba C, Bucarey JL, Tapia G, Valenzuela R, Contreras-Ferrat A, Llanos P, Jaimovich E.
    Insulin-dependent H2O2 production is higher in muscle fibers of mice fed with a high-fat diet.
    Int J Mol Sci. 2013 Jul 29;14(8):15740-54 doi: 10.3390/ijms140815740
    pmid: 23899788
  1. Insulin-resistant mice have increased H2O2 release upon insulin stimulation. Effect was mediated by an increased NOX2 expression.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: Mouse skeletal muscle fibers.
  4. Detection system: Olympus IX81-DSU Spinning Disk Confocal Microscope.
  • Matus S, Lopez E, Valenzuela V, Nassif M, Hetz C.
    Functional contribution of the transcription factor ATF4 to the pathogenesis of amyotrophic lateral sclerosis.
    PLoS One. 2013 Jul 18;8(7):e66672 doi: 10.1371/journal.pone.0066672
    pmid: 23874395
     
  • Sun Q, Gao W, Loughran P, Shapiro R, Fan J, Billiar TR, Scott MJ.
    Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.
    J Biol Chem. 2013 May 31;288(22):15947-58 doi: 10.1074/jbc.M112.426791
    pmid: 23589298
  1. Constructs used: pHyPer-cyto and pHyPer-dMito vectors.
  2. Expression system: Mouse hepatocytes.
  3. Detection system: EVOS fluorescence microscope (AMG).
  • Martinelli R, Kamei M, Sage PT, Massol R, Varghese L, Sciuto T, Toporsian M, Dvorak AM, Kirchhausen T, Springer TA, Carman CV.
    Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.
    J Cell Biol. 2013 Apr 29;201(3):449-65 doi: 10.1083/jcb.201209077
    pmid: 23629967
     
  • Hajas G, Bacsi A, Aguilera-Aguirre L, Hegde ML, Tapas KH, Sur S, Radak Z, Ba X, Boldogh I.
    8-Oxoguanine DNA glycosylase-1 links DNA repair to cellular signaling via the activation of the small GTPase Rac1.
    Free Radic Biol Med. 2013 Apr 21;61C:384-394 doi: 10.1016/j.freeradbiomed.2013.04.011
    pmid: 23612479
  1. A product of DNA base lesion 8-oxoG increases NOX4 mediated ROS production. Using targeted versions of HyPer it is shown that ROS production is colocalized with NOX4 and Rac1 on a nuclear membrane.
  2. Constructs used: pHyPer-cyto, pHyPer-dMito, pHyPer-nuc vectors.
  3. Expression system: A549 typeII alveolar epithelial cells and human embryonic fibroblasts MRC-5.
  4. Detection system: TCS-SP2 confocal microscope.
  • Kim E, Gordonov T, Liu Y, Bentley WE, Payne GF.
    Reverse engineering to suggest biologically relevant redox activities of phenolic materials.
    ACS Chem Biol. 2013 Apr 19;8(4):716-24 doi: 10.1021/cb300605s
    pmid: 23320381
  1. Constructs used: HyPer cloned into pQE30 vector.
  2. Expression system: E. coli.
  3. Detection system: Microplate reader.
  • Birk J, Meyer M, Aller I, Hansen HG, Odermatt A, Dick TP, Meyer AJ, Appenzeller-Herzog C.
    Endoplasmic reticulum: reduced and oxidized glutathione revisited.
    J Cell Sci. 2013 Apr 1;126(Pt 7):1604-17 doi: 10.1242/jcs.117218
    pmid: 23424194
  1. Constructs used: HyPer, targeted to the endoplasmic reticulum.
  2. Expression system: HeLa cells.
  3. Detection system: Live cell imaging, Olympus Fluoview 1000 laser scanning confocal microscope, 410–510 nm excitation range at 535 nm emission.
  • Andrio E, Marino D, Marmeys A, de Segonzac MD, Damiani I, Genre A, Huguet S, Frendo P, Puppo A, Pauly N.
    Hydrogen peroxide-regulated genes in the Medicago truncatula-Sinorhizobium meliloti symbiosis.
    New Phytol. 2013 Apr;198(1):179-89 doi: 10.1111/nph.12120
    pmid: 23347006
  1. Hydrogen peroxide production level correlates with a protein kinase MtSpk1 expression in plant roots. ROS regulates expression of genes, necessary for regulation of Medicago truncatula–Sinorhizobium meliloti symbiotic interaction.
  2. Constructs used: HyPer, transferred to the binary plasmid pK2GW7.
  3. Expression system: Medicago truncatula plants.
  4. Detection system: TCS-SP2 confocal microscope.
  • Panieri E, Gogvadze V, Norberg E, Venkatesh R, Orrenius S, Zhivotovsky B.
    Reactive oxygen species generated in different compartments induce cell death, survival, or senescence.
    Free Radic Biol Med. 2013 Apr;57:176-87 doi: 10.1016/j.freeradbiomed.2012.12.024
    pmid: 23295411
  1. Constructs used: pHyPer-dMito, pHyPer-Cyto or pHyPer-Nuc constructs.
  2. Expression system: U1810 and A549 human non-small cell lung carcinoma cell lines.
  3. Detection system: Zeiss LSM-510-META confocal microscope equipped with a Plan Neofluar 25X/0.8 lmm corr DIC objective and 30mW Argon/2 laser. HyPer fluorescence was acquired using 488nm excitation laser line (6% intensity) and detected at 505-550nm wavelength.
  • Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella TW, Grabher C, Schultz C, Lukyanov S, Belousov VV.
    HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging.
    ACS Chem Biol. 2013 Mar 15;8(3):535-42 doi: 10.1021/cb300625g
    pmid: 23256573
  1. Constructs used: pC1-HyPer3.
  2. Expression system: 1) Hela, transfected with Fugene6;
    2) Zebrafish, transgenesis performed by co-injection of vector DNA plus tol2 mRNA. Resulting transgenics exhibited a ubiquitous expression (b-actin promoter) of HyPer-3.
  3. Detection system: 1) Leica DMI 6000 B. For the channel corresponding to the protonated form of the cpYFP chromophore (420 nm), following filters were used: excitation: 427/10 (CFP), emission: BP 542/27 (YFP). For the deprotonated form of the cpYFP chromophore (500 nm): excitation BP 504/12 (YFP), emission: BP 542/27 (YFP).
    2) Frequency-domain FLIM measurements were performed using LIFA FLIM setup on an Olympus 71 inverted microscope equipped with a Olympus 30x NA 1.05 lens. Excitation was 488 nm, emission in the GFP range. Reference was Atto488.
  • Gibbs-Flournoy EA, Simmons SO, Bromberg PA, Dick TP, Samet JM.
    Monitoring intracellular redox changes in ozone-exposed airway epithelial cells.
    Environ Health Perspect. 2013 Mar;121(3):312-7 doi: 10.1289/ehp.1206039
    pmid: 23249900
  1. Measuring H2O2 production in airway epithelial cells, exposed to O3.
  2. Constructs used: pHyPer-cyto and pHyPer-dMito vectors.
  3. Expression system: BEAS-2B Human epithelial cells.
  4. Detection system: Nikon Eclipse C1si spectral confocal imaging system equipped with an Eclipse Ti microscope, Perfect Focus System, and 404 nm, 488 nm, 561 nm, and 633 nm primary laser lines. Images were acquired using a 60× Plan Apo lens. Green fluorescence was observed via the use of independent excitations at 404 and 488 nm, and emitted light was collected for each using a 525/30 nm band-pass filter.
  • Gandhirajan RK, Meng S, Chandramoorthy HC, Mallilankaraman K, Mancarella S, Gao H, Razmpour R, Yang XF, Houser SR, Chen J, Koch WJ, Wang H, Soboloff J, Gill DL, Madesh M.
    Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation.
    J Clin Invest. 2013 Feb 1;123(2):887-902 doi: 10.1172/JCI65647
    pmid: 23348743
  1. Expression system: MPMVECs stably expressing HyPer-Cyto.
  2. Detection system: Zeiss LSM510 using a ×40/1.3 NA oil objective at room temperature and using a 488-nm laser at 2.5% power.
  • Brynildsen MP, Winkler JA, Spina CS, MacDonald IC, Collins JJ.
    Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production.
    Nat Biotechnol. 2013 Feb;31(2):160-5 doi: 10.1038/nbt.2458
    pmid: 23292609
  1. Constructs used: HyPer, cloned into pQE30.
  2. Expression system: Escherichia coli MG1655 strain.
  3. Detection system: SpectraMax M5 plate reader, excitation: 420 nm and 500 nm, emission: 530 nm, bottom read.
  • Love NR, Chen Y, Ishibashi S, Kritsiligkou P, Lea R, Koh Y, Gallop JL, Dorey K, Amaya E.
    Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration.
    Nat Cell Biol. 2013 Feb;15(2):222-8 doi: 10.1038/ncb2659
    pmid: 23314862
  1. Expression system: Xenopus laevis transgenic lines that express HyPer ubiquitously from the CMV promoter.
  2. Detection system: For live imaging, specimens were anaethesized in a filter sterilized solution of 0.1X MMR solution containing 0.025% MS222 (pH 7.5) and placed in a glass bottom dish. HyPer and pHluorin ratiometric imaging analysis was performed using a Nikon TE2000 PFS microscope, a mercury illumination source, and a Cascade II EMCCD camera (Photometric) using excitation filters of 402/15 nm and 490/20 nm, and an emission filter of BP525/36.
  • Barata AG, Dick TP.
    In vivo imaging of H2O2 production in Drosophila.
    Methods Enzymol. 2013;526:61-82 doi: 10.1016/B978-0-12-405883-5.00004-1
    pmid: 23791094
     
  • Bonekamp NA, Islinger M, Lázaro MG, Schrader M.
    Cytochemical detection of peroxisomes and mitochondria.
    Methods Mol Biol. 2013;931:467-82 doi: 10.1007/978-1-62703-056-4_24
    pmid: 23027018
  1. Protocols for dual peroxisomes and mitochondria staining.
  • Enyedi B, Niethammer P.
    H2O2: a chemoattractant?
    Methods Enzymol. 2013;528:237-55 doi: 10.1016/B978-0-12-405881-1.00014-8
    pmid: 23849869
     
  • Exposito-Rodriguez M, Laissue PP, Littlejohn GR, Smirnoff N, Mullineaux PM.
    The use of HyPer to examine spatial and temporal changes in H2O2 in high light-exposed plants.
    Methods Enzymol. 2013;527:185-201 doi: 10.1016/B978-0-12-405882-8.00010-6
    pmid: 23830632
     
  • Hernández-Barrera A, Quinto C, Johnson EA, Wu HM, Cheung AY, Cárdenas L.
    Using hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: a method with virtually unlimited potential in plant biology.
    Methods Enzymol. 2013;527:275-90 doi: 10.1016/B978-0-12-405882-8.00015-5
    pmid: 23830637
  1. Protocol, describing HyPer transfection and imaging in Arabidopsis.
  2. Constructs used: HyPer, cloned into a Ti plasmid-based vector behind either the constitutively active CaMV35S promoter or the pollen specific promoter LAT52.
  3. Expression system: Arabidopsis thaliana plants, transfected by floral dip method.
  4. Detection system: All images were acquired with a CCD camera attached to a Nikon TE300 inverted microscope coupled to a xenon illumination source DG-4, which contains a 175-W ozone-free xenon lamp (330–700 nm) and a galvanometer-driven wavelength switcher.
  • Kniss A, Lu H, Jones DP, Kemp ML.
    A microfluidic systems biology approach for live single-cell mitochondrial ROS imaging.
    Methods Enzymol. 2013;526:219-30 doi: 10.1016/B978-0-12-405883-5.00013-2
    pmid: 23791103
     
  • Mishina NM, Markvicheva KN, Bilan DS, Matlashov ME, Shirmanova MV, Liebl D, Schultz C, Lukyanov S, Belousov VV.
    Visualization of intracellular hydrogen peroxide with HyPer, a genetically encoded fluorescent probe.
    Methods Enzymol. 2013;526:45-59 doi: 10.1016/B978-0-12-405883-5.00003-X
    pmid: 23791093
  1. Protocol
  • Mishina NM, Markvicheva KN, Fradkov AF, Zagaynova EV, Schultz C, Lukyanov S, Belousov VV.
    Imaging H2O2 microdomains in receptor tyrosine kinases signaling.
    Methods Enzymol. 2013;526:175-87 doi: 10.1016/B978-0-12-405883-5.00011-9
    pmid: 23791101
  1. Protocol
  • Sartoretto JL, Kalwa H, Romero N, Michel T.
    In vivo imaging of nitric oxide and hydrogen peroxide in cardiac myocytes.
    Methods Enzymol. 2013;528:61-78 doi: 10.1016/B978-0-12-405881-1.00004-5
    pmid: 23849859
     
  • Mishina NM, Bogeski I, Bolotin DA, Hoth M, Niemeyer BA, Schultz C, Zagaynova EV, Lukyanov S, Belousov VV.
    Can we see PIP(3) and hydrogen peroxide with a single probe?
    Antioxid Redox Signal. 2012 Aug 1;17(3):505-12 doi: 10.1089/ars.2012.4574
    pmid: 22369174
  1. HyPer-Btk fusion protein was used as a new sensor PIP-SHOW for a simultaneous measurement of H2O2 production and PIP3 kinase activity.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: Hela-Kyoto and NIH-3T3 cells.
  4. Detection system: Zeiss Cell Observer HS widefield microscope equipped with a 40x Fluar oil lense (N.A.1.3) and CFP, YFP dualband pinkel filter set (55HE, Zeiss); fluorescence was detected using excitation at 420 and 505 nm and emission at 515 nm. Leica 6000 widefield microscope equipped with a 20x air objective and HCX PL APO lbd.BL 63 x 1.4NA oil objective; fluorescence was excited sequentially using 427/10 and 504/12 band-pass excitation filters, emission was collected using a 525/50 bandpass emission filter.
  • Godfrey R, Arora D, Bauer R, Stopp S, Müller JP, Heinrich T, Böhmer SA, Dagnell M, Schnetzke U, Scholl S, Östman A, Böhmer FD.
    Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ.
    Blood. 2012 May 10;119(19):4499-511 doi: 10.1182/blood-2011-02-336446
    pmid: 22438257
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: HEK293 cell line.
  3. Detection system: Zeiss LSM 510 confocal microscope.
  • Iida R, Ueki M, Yasuda T.
    Identification of Rhit as a novel transcriptional repressor of human Mpv17-like protein with a mitigating effect on mitochondrial dysfunction, and its transcriptional regulation by FOXD3 and GABP.
    Free Radic Biol Med. 2012 Apr 15;52(8):1413-22 doi: 10.1016/j.freeradbiomed.2012.01.003
    pmid: 22306510
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: MCF7 cell line.
  3. Detection system: Multilabel reader, ex/em = 490/535 nm.
  • Choi WG, Swanson SJ, Gilroy S.
    High-resolution imaging of Ca2+ , redox status, ROS and pH using GFP biosensors.
    Plant J. 2012 Apr;70(1):118-28 doi: 10.1111/j.1365-313X.2012.04917.x
    pmid: 22449047
  1. Review
  • Back P, De Vos WH, Depuydt GG, Matthijssens F, Vanfleteren JR, Braeckman BP.
    Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans.
    Free Radic Biol Med. 2012 Mar 1;52(5):850-9 doi: 10.1016/j.freeradbiomed.2011.11.037
    pmid: 22226831
  1. Transgenic C. elegans line expressing HyPer was generated. Local "hot spots" of H2O2 production in an intact worm were detected. Hydrogen peroxide level increased with age and was delayed by dietary restriction.
  2. Constructs used: HyPer, integrated into expression vector with the ribosomal rpl-17 promoter.
  3. Expression system: Caenorhabditis elegans worms, transfected with the Biolistic PDS-1000/He particle delivery system.
  4. Detection system: Victor2 1420 multilabel counter with 490 nm and 405 nm excitation filters and a 535 nm emission filter. Also Nikon A1r confocal laser scanning microscopy system mounted on a Nikon Ti-E inverted epifluorescence microscope; fluorescence excited with a 405 nm diode laser and 488 nm multiline Ar laser and detected through a 525/50 nm bandpass filter.
  • Gusdon AM, Zhu J, Van Houten B, Chu CT.
    ATP13A2 regulates mitochondrial bioenergetics through macroautophagy.
    Neurobiol Dis. 2012 Mar;45(3):962-72 doi: 10.1016/j.nbd.2011.12.015
    pmid: 22198378
  1. Elevated ROS production in neuronal mitochondria in Parkinson disease model. Used as an alternative to MitoSOX Red.
  2. Constructs used: pHyPer-cyto vector, pHyPer-dMito vector.
  3. Expression system: Mouse primary cortical neurons and SH-SY5Y cell line.
  4. Detection system: SpectraMax A2 plate reader (Molecular Devices, Sunnyvale, CA), ex/em = 500/516 nm.
  • Cheng WY, Currier J, Bromberg PA, Silbajoris R, Simmons SO, Samet JM.
    Linking oxidative events to inflammatory and adaptive gene expression induced by exposure to an organic particulate matter component.
    Environ Health Perspect. 2012 Feb;120(2):267-74 doi: 10.1289/ehp.1104055
    pmid: 21997482
  1. An environmental toxin 1,2-NQ exposure induced mitochondrial production of H2O2 that mediated the expression of inflammatory genes.
  2. Constructs used: HyPer, cloned into the lentiviral transfer vector pTLRED.
  3. Expression system: BEAS-2B human airway epithelial cells, lentiviral transduction.
  4. Detection system: Nikon C1Si system equipped with an Eclipse Ti microscope, 404 and 488 nm laser lines and 525/50 nm band-pass emission filter.
  • Yoshii H, Yoshii Y, Asai T, Furukawa T, Takaichi S, Fujibayashi Y.
    Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production.
    Biochem Biophys Res Commun. 2012 Jan 6;417(1):640-5 doi: 10.1016/j.bbrc.2011.12.024
    pmid: 22185691
  1. Measuring ROS, produced by photo-excited carotenoids.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: HeLa cell line, stable expression.
  4. Detection system: Olympus IX70 fluorescent microscope.
  • Feine I, Pinkas I, Salomon Y, Scherz A.
    Local oxidative stress expansion through endothelial cells--a key role for gap junction intercellular communication.
    PLoS One. 2012;7(7):e41633 doi: 10.1371/journal.pone.0041633
    pmid: 22911831
  1. Measuring H2O2 production in endothelial cells during propagation of oxidative insult. ROS elevation precedes cellular death.
  2. Constructs used: pHyPer-cyto vector.
  3. Expression system: H5V mouse heart endothelial cells, transient expression.
  4. Detection system: Olympus BX61 microscope, equipped with Lambda DG-4 illumination system, Cascade 512B EM-CCD. λex = 420 and 490 nm and λem = 520 nm.
  • Pase L, Nowell CJ, Lieschke GJ.
    In vivo real-time visualization of leukocytes and intracellular hydrogen peroxide levels during a zebrafish acute inflammation assay.
    Methods Enzymol. 2012;506:135-56 doi: 10.1016/B978-0-12-391856-7.00032-9
    pmid: 22341223
  1. Protocol for in-vivo imaging in Zebrafish.
  • 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 doi: 10.1073/pnas.1111331108
    pmid: 21896719
  1. Constructs used: HyPer2.
  2. Expression system: Mouse cardiac myocytes.
  3. Detection system: HyPer2 fluorescence was excited with 420∕40 and with 500∕16 band-pass excitation filters; corresponding YFP emission was acquired every 5 s for 10 min using a 535∕30 band-pass emission filter.
  • 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 doi: 10.1126/scisignal.2001127
    pmid: 21521879
  1. Constructs used: pHyPer-cyto.
  2. Expression system: DRG neurons.
  3. Detection system: Olympus IX81 microscope equipped with an Olympus DP30BW camera. GFP-HyPer was excited at 525 nm, and fluorescence from GHP-HyPer was monitored at 480 nm.
  • 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 doi: 10.2337/db09-1401
    pmid: 20971967
  1. Constructs used: HyPer-dMito, HyPer-peroxi.
  2. Expression system: RINm5F cells, primary rat islet cells.
  3. Detection system: CellR Olympus IX 81 inverted microscope system.
  • Enyedi B, Várnai P, Geiszt M.
    Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium.
    Antioxid Redox Signal. 2010 Sep 15;13(6):721-9 doi: 10.1089/ars.2009.2880
    pmid: 20095866
  1. Constructs used: HyPer-cyto, HyPer-dMito, HyPer-ER, HyPer-3NLS, HyPer-mem.
  2. Expression system: Hela.
  3. Detection system: Confocal images were collected on a Zeiss LSM510 confocal laser scanning microscope, equipped with a 63x1.4 oil immersion objective on a thermostated stage. HyPer was excited with 488-nm argon, emission was collected by using a 500- to 530-nm bandpass filter.
  • 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 doi: 10.1073/pnas.1005776107
    pmid: 20724658
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: Transiently transfected HEK 293 cells and HeLa cells.
  3. Detection system: 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 470/40 nm, a beam splitter at 495 nm, and a bandpass emission filter at 525/50 nm.
  • 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 doi: 10.1161/CIRCRESAHA.110.224345
    pmid: 20616314
  1. Constructs used: pHyPer-cyto vector.
  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 doi: 10.1128/MCB.01445-09
    pmid: 20457808
  1. Constructs used: pHyPer-cyto vector, pHyPer-dMito vector, ER-targeted HyPer.
  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 doi: 10.1158/0008-5472.CAN-09-4336
    pmid: 20424115
  1. Constructs used: pHyPer-cyto and pHyPer-nuc vectors.
  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 doi: 10.1111/j.1365-313X.2010.04190.x
    pmid: 20230493
  1. Constructs used: 1) HyPer under the control of double 35S promoter and TEV translational enhancer;
    2) HyPer targeted to peroxisomes.
  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 doi: 10.1152/ajpheart.00375.2009
    pmid: 19749157
  1. Constructs used: pHyPer-dMito vector.
  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 doi: 10.1073/pnas.0907409106
    pmid: 19805165
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: transiently transfected bovine aortic endothelial cells (BAEC).
  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 doi: 10.1038/nature08119
    pmid: 19494811
  1. Constructs used: HyPer mRNA.
  2. Expression system: Zebrafish embryos.
  3. Detection system: Inverted widefield microscope equipped with a CCD camera and a mercury illumination source.
  • 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 doi: 10.1007/s00403-009-0935-9
    pmid: 19306099
  1. Constructs used: HyPer and HyPer-dMito subcloned into adenoviral vectors.
  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 doi: 10.1074/jbc.M804249200
    pmid: 19028699
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: Primary culture of mouse skeletal muscle cells.
  3. Detection system: Zeiss LSM-5, Pascal 5 Axiovert 200 microscope.
     
  • 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 doi: 10.1152/physrev.00031.2007
    pmid: 18923182
  1. Review
  • 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. 2008;476:79-86
    pmid: 19157010
  1. Constructs used: pHyPer-cyto vector.
  2. Expression system: transiently transfected HeLa cells.
  3. Detection system: Leica confocal system TCS SP2 (excitation for laser 488 nm, 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.
  • Souslova EA, Chudakov DM.
    Genetically encoded intracellular sensors based on fluorescent proteins.
    Biochemistry (Mosc). 2007 Jul;72(7):683-97
    pmid: 17680759
  1. Review
  • 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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