Measuring Calcium and ROS by Genetically Encoded Protein Sensors and Fluorescent Dyes

  • Christine S. Gibhardt
  • Adina Vultur
  • Ivan BogeskiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)


Oxidative modifications of cellular building blocks such as proteins, lipids, and DNA have a major impact on cell behavior, fate, and clinical outcome. Reactive oxygen species (ROS) are important factors that influence these redox processes. Calcium ion (Ca2+) dynamics and signals are also essential regulators of key cellular processes. Therefore, the combined and precise monitoring of ROS and Ca2+ in single cells, with a high spatial and temporal resolution and in physiological environments, is essential to better understand their functional impact. Here, we describe protocols to detect one of the most prominent ROS (hydrogen peroxide, H2O2) using genetically encoded protein sensors and fluorescent dyes. We also provide guidelines on how to simultaneously detect Ca2+ and H2O2 and how to examine the influence of Ca2+ signals on cellular ROS production and vice versa.

Key words

Calcium ROS Redox Imaging Microscopy Sensor H2O2 


  1. 1.
    Storz P (2005) Reactive oxygen species in tumor progression. Front Biosci 10:1881–1896CrossRefGoogle Scholar
  2. 2.
    Le Gal K, Ibrahim MX, Wiel C, Sayin VI, Akula MK, Karlsson C, Dalin MG, Akyurek LM, Lindahl P, Nilsson J, Bergo MO (2015) Antioxidants can increase melanoma metastasis in mice. Sci Transl Med 7(308):308re8CrossRefGoogle Scholar
  3. 3.
    Sayin VI, Ibrahim MX, Larsson E, Nilsson JA, Lindahl P, Bergo MO (2014) Antioxidants accelerate lung cancer progression in mice. Sci Transl Med 6(221):221ra15CrossRefGoogle Scholar
  4. 4.
    Piskounova E, Agathocleous M, Murphy MM, Hu Z, Huddlestun SE, Zhao Z, Leitch AM, Johnson TM, DeBerardinis RJ, Morrison SJ (2015) Oxidative stress inhibits distant metastasis by human melanoma cells. Nature 527(7577):186–191CrossRefGoogle Scholar
  5. 5.
    Bogeski I, Kappl R, Kummerow C, Gulaboski R, Hoth M, Niemeyer BA (2011) Redox regulation of calcium ion channels: chemical and physiological aspects. Cell Calcium 50(5):407–423CrossRefGoogle Scholar
  6. 6.
    Murphy MP, Holmgren A, Larsson NG, Halliwell B, Chang CJ, Kalyanaraman B, Rhee SG, Thornalley PJ, Partridge L, Gems D, Nystrom T, Belousov V, Schumacker PT, Winterbourn CC (2011) Unraveling the biological roles of reactive oxygen species. Cell Metab 13(4):361–366CrossRefGoogle Scholar
  7. 7.
    Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82(1):47–95CrossRefGoogle Scholar
  8. 8.
    D'Autreaux B, Toledano MB (2007) ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8(10):813–824CrossRefGoogle Scholar
  9. 9.
    Diebold L, Chandel NS (2016) Mitochondrial ROS regulation of proliferating cells. Free Radic Biol Med 100:86–93CrossRefGoogle Scholar
  10. 10.
    Sena LA, Chandel NS (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48(2):158–167CrossRefGoogle Scholar
  11. 11.
    Clapham DE (2007) Calcium signaling. Cell 131(6):1047–1058CrossRefGoogle Scholar
  12. 12.
    Hempel N, Trebak M (2017) Crosstalk between calcium and reactive oxygen species signaling in cancer. Cell Calcium 63:70–96CrossRefGoogle Scholar
  13. 13.
    Hoth M (2016) CRAC channels, calcium, and cancer in light of the driver and passenger concept. Biochim Biophys Acta 1863(6 Pt B):1408–1417CrossRefGoogle Scholar
  14. 14.
    Tosatto A, Sommaggio R, Kummerow C, Bentham RB, Blacker TS, Berecz T, Duchen MR, Rosato A, Bogeski I, Szabadkai G, Rizzuto R, Mammucari C (2016) The mitochondrial calcium uniporter regulates breast cancer progression via HIF-1alpha. EMBO Mol Med 8(5):569–585CrossRefGoogle Scholar
  15. 15.
    Bogeski I, Niemeyer BA (2014) Redox regulation of ion channels. Antioxid Redox Signal 21(6):859–862CrossRefGoogle Scholar
  16. 16.
    Booth DM, Enyedi B, Geiszt M, Varnai P, Hajnoczky G (2016) Redox nanodomains are induced by and control calcium signaling at the ER-mitochondrial interface. Mol Cell 63(2):240–248CrossRefGoogle Scholar
  17. 17.
    Bertero E, Maack C (2018) Calcium signaling and reactive oxygen species in mitochondria. Circ Res 122(10):1460–1478CrossRefGoogle Scholar
  18. 18.
    Saul S, Gibhardt CS, Schmidt B, Lis A, Pasieka B, Conrad D, Jung P, Gaupp R, Wonnenberg B, Diler E, Stanisz H, Vogt T, Schwarz EC, Bischoff M, Herrmann M, Tschernig T, Kappl R, Rieger H, Niemeyer BA, Bogeski I (2016) A calcium-redox feedback loop controls human monocyte immune responses: the role of ORAI Ca2+ channels. Sci Signal 9(418):ra26CrossRefGoogle Scholar
  19. 19.
    Bogeski I, Kummerow C, Al-Ansary D, Schwarz EC, Koehler R, Kozai D, Takahashi N, Peinelt C, Griesemer D, Bozem M, Mori Y, Hoth M, Niemeyer BA (2010) Differential redox regulation of ORAI ion channels: a mechanism to tune cellular calcium signaling. Sci Signal 3(115):ra24CrossRefGoogle Scholar
  20. 20.
    Gibhardt CS, Zimmermann KM, Zhang X, Belousov VV, Bogeski I (2016) Imaging calcium and redox signals using genetically encoded fluorescent indicators. Cell Calcium 60(2):55–64CrossRefGoogle Scholar
  21. 21.
    Rezende F, Brandes RP, Schroder K (2017) Detection of hydrogen peroxide with fluorescent dyes. Antioxid Redox Signal 29:585–602CrossRefGoogle Scholar
  22. 22.
    Swain L, Nanadikar MS, Borowik S, Zieseniss A, Katschinski DM (2018) Transgenic organisms meet redox bioimaging: one step closer to physiology. Antioxid Redox Signal 29:603–612CrossRefGoogle Scholar
  23. 23.
    Bozem M, Knapp P, Mirceski V, Slowik EJ, Bogeski I, Kappl R, Heinemann C, Hoth M (2017) Electrochemical quantification of extracellular local H2O2 kinetics originating from single cells. Antioxid Redox Signal 29:501–517CrossRefGoogle Scholar
  24. 24.
    Roma LP, Deponte M, Riemer J, Morgan B (2018) Mechanisms and applications of redox- sensitive green fluorescent protein-based hydrogen peroxide probes. Antioxid Redox Signal 29:552–568CrossRefGoogle Scholar
  25. 25.
    Bilan D, Belousov V (2018) In vivo imaging of hydrogen peroxide with HyPer probes. Antioxid Redox Signal 29:569–584CrossRefGoogle Scholar
  26. 26.
    Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4):281–286CrossRefGoogle Scholar
  27. 27.
    Gutscher M, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, Samstag Y, Dick TP (2009) Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. J Biol Chem 284(46):31532–31540CrossRefGoogle Scholar
  28. 28.
    Markvicheva KN, Bilan DS, Mishina NM, Gorokhovatsky AY, Vinokurov LM, Lukyanov S, Belousov VV (2011) A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg Med Chem 19(3):1079–1084CrossRefGoogle Scholar
  29. 29.
    Bilan DS, Pase L, Joosen L, Gorokhovatsky AY, Ermakova YG, Gadella TWJ, Grabher C, Schultz C, Lukyanov S, Belousov VV (2013) HyPer-3: a genetically encoded H2O2 probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem Biol 8(3):535–542CrossRefGoogle Scholar
  30. 30.
    Ermakova YG, Bilan DS, Matlashov ME, Mishina NM, Markvicheva KN, Subach OM, Subach FV, Bogeski I, Hoth M, Enikolopov G, Belousov VV (2014) Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. Nat Commun 5:5222CrossRefGoogle Scholar
  31. 31.
    Mishina NM, Bogeski I, Bolotin DA, Hoth M, Niemeyer BA, Schultz C, Zagaynova EV, Lukyanov S, Belousov VV (2012) Can we see PIP3 and hydrogen peroxide with a single probe? Antioxid Redox Signal 17(3):505–512CrossRefGoogle Scholar
  32. 32.
    Morgan B, Van Laer K, Owusu TNE, Ezerina D, Pastor-Flores D, Amponsah PS, Tursch A, Dick TP (2016) Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. Nat Chem Biol 12(6):437–443CrossRefGoogle Scholar
  33. 33.
    Lin MZ, Schnitzer MJ (2016) Genetically encoded indicators of neuronal activity. Nat Neurosci 19(9):1142–1153CrossRefGoogle Scholar
  34. 34.
    Pérez KV, Nagai T (2013) Genetically encoded Ca2+ indicators: Properties and evaluation. Biochim Biophys Acta 1833(7):1787–1797CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Christine S. Gibhardt
    • 1
  • Adina Vultur
    • 1
  • Ivan Bogeski
    • 1
    Email author
  1. 1.Molecular Physiology, Institute of Cardiovascular PhysiologyUniversity Medical Center, Georg-August-UniversityGöttingenGermany

Personalised recommendations