Cellular oxidation–reduction reactions are mainly regulated by pyridine nucleotides (NADPH/NADP+ and NADH/NAD+), thiols, and reactive oxygen species (ROS) and play central roles in cell metabolism, cellular signaling, and cell-fate decisions. A comprehensive evaluation or multiplex analysis of redox landscapes and dynamics in intact living cells is important for interrogating cell functions in both healthy and disease states; however, until recently, this goal has been limited by the lack of a complete set of redox sensors. We recently reported the development of a series of highly responsive, genetically encoded fluorescent sensors for NADPH that substantially strengthen the existing toolset of genetically encoded sensors for thiols, H2O2, and NADH redox states. By combining sensors with unique spectral properties and specific subcellular targeting domains, our approach allows simultaneous imaging of up to four different sensors. In this protocol, we first describe strategies for multiplex fluorescence imaging of these sensors in single cells; then we demonstrate how to apply these sensors to study changes in redox landscapes during the cell cycle, after macrophage activation, and in living zebrafish. This approach can be adapted to different genetically encoded fluorescent sensors and various analytical platforms such as fluorescence microscopy, high-content imaging systems, flow cytometry, and microplate readers. A typical preparation of cells or zebrafish expressing different sensors takes 2–3 d; microscopy imaging or flow-cytometry analysis can be performed within 5–60 min.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Zhao, Y., Zhang, Z., Zou, Y. & Yang, Y. Visualization of nicotine adenine dinucleotide redox homeostasis with genetically encoded fluorescent sensors. Antioxid. Redox Signal. 28, 213–229 (2018).
Fan, J. et al. Quantitative flux analysis reveals folate-dependent NADPH production. Nature 510, 298–302 (2014).
Cairns, R. A., Harris, I. S. & Mak, T. W. Regulation of cancer cell metabolism. Nat. Rev. Cancer 11, 85–95 (2011).
Gorrini, C., Harris, I. S. & Mak, T. W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 12, 931–947 (2013).
Ying, W. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid. Redox Signal. 10, 179–206 (2008).
Blacker, T. S. et al. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat. Commun. 5, 3936 (2014).
Maddocks, O. D., Labuschagne, C. F. & Vousden, K. H. Localization of NADPH production: a wheel within a wheel. Mol. Cell 55, 158–160 (2014).
Tao, R. et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. Nat. Methods 14, 720–728 (2017).
Veech, R. L., Eggleston, L. V. & Krebs, H. A. The redox state of free nicotinamide-adenine dinucleotide phosphate in the cytoplasm of rat liver. Biochem. J. 115, 609–619 (1969).
Canto, C. et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458, 1056–1060 (2009).
Wise, D. D. & Shear, J. B. Tracking variations in nicotinamide cofactors extracted from cultured cells using capillary electrophoresis with multiphoton excitation of fluorescence. Anal. Biochem. 326, 225–233 (2004).
Lewis, C. A. et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol. Cell 55, 253–263 (2014).
Quinn, K. P. et al. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation. Sci. Rep. 3, 3432 (2013).
Hanson, G. T. et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J. Biol. Chem. 279, 13044–13053 (2004).
Dooley, C. T. et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279, 22284–22293 (2004).
Gutscher, M. et al. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5, 553–559 (2008).
Fan, Y., Chen, Z. & Ai, H. W. Monitoring redox dynamics in living cells with a redox-sensitive red fluorescent protein. Anal. Chem. 87, 2802–2810 (2015).
Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).
Ermakova, Y. G. et al. Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. Nat. Commun. 5, 5222 (2014).
Albrecht, S. C., Barata, A. G., Grosshans, J., Teleman, A. A. & Dick, T. P. In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab. 14, 819–829 (2011).
Morgan, B. et al. Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. Nat. Chem. Biol. 12, 437–443 (2016).
Zhao, Y. et al. SoNar, a highly responsive NAD+/NADH sensor, allows high-throughput metabolic screening of anti-tumor agents. Cell Metab. 21, 777–789 (2015).
Zhao, Y. et al. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state. Nat. Protoc. 11, 1345–1359 (2016).
Zhao, Y. et al. Genetically encoded fluorescent sensors for intracellular NADH detection. Cell Metab. 14, 555–566 (2011).
Wiederkehr, A. & Demaurex, N. Illuminating redox biology using NADH- and NADPH-specific sensors. Nat. Methods 14, 671–672 (2017).
Zhao, Y. & Yang, Y. Profiling metabolic states with genetically encoded fluorescent biosensors for NADH. Curr. Opin. Biotechnol. 31C, 86–92 (2015).
Hung, Y. P., Albeck, J. G., Tantama, M. & Yellen, G. Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor. Cell Metab. 14, 545–554 (2011).
Bilan, D. S. et al. Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments. Biochim. Biophys. Acta 1840, 951–957 (2014).
Zhao, Y. & Yang, Y. Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD+/NADH sensors. Free Radic. Biol. Med. 100, 43–52 (2016).
Zhang, J. et al. Determination of the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae using shikimate dehydrogenase as sensor reaction. Sci. Rep. 5, 12846 (2015).
Hedeskov, C. J., Capito, K. & Thams, P. Cytosolic ratios of free [NADPH]/[NADP+] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion. Biochem. J. 241, 161–167 (1987).
Zhang, Q., Piston, D. W. & Goodman, R. H. Regulation of corepressor function by nuclear NADH. Science 295, 1895–1897 (2002).
Mayevsky, A. & Rogatsky, G. G. Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am. J. Physiol. Cell Physiol. 292, C615–C640 (2007).
Murphy, M. P. et al. Unraveling the biological roles of reactive oxygen species. Cell Metab. 13, 361–366 (2011).
Pollak, N., Niere, M. & Ziegler, M. NAD kinase levels control the NADPH concentration in human cells. J. Biol. Chem. 282, 33562–33571 (2007).
Ohashi, K., Kawai, S. & Murata, K. Identification and characterization of a human mitochondrial NAD kinase. Nat. Commun. 3, 1248 (2012).
Cambronne, X. A. et al. Biosensor reveals multiple sources for mitochondrial NAD(+). Science 352, 1474–1477 (2016).
Cameron, W. D. et al. Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+). Nat. Methods 13, 352–358 (2016).
Hoek, J. B. & Rydstrom, J. Physiological roles of nicotinamide nucleotide transhydrogenase. Biochem. J. 254, 1–10 (1988).
Zhang, X., Edwards, J. P. & Mosser, D. M. The expression of exogenous genes in macrophages: obstacles and opportunities. Methods Mol. Biol. 531, 123–143 (2009).
Ghesquiere, B., Wong, B. W., Kuchnio, A. & Carmeliet, P. Metabolism of stromal and immune cells in health and disease. Nature 511, 167–176 (2014).
Karlsson, J., von Hofsten, J. & Olsson, P.E. Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar. Biotechnol. (NY) 3, 522–527 (2001).
Villalobos, A., Ness, J. E., Gustafsson, C., Minshull, J. & Govindarajan, S. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics 7, 285 (2006).
Schwartz, M. A., Schaller, M. D. & Ginsberg, M. H. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11, 549–599 (1995).
Hynes, R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25 (1992).
Tiscornia, G., Singer, O. & Verma, I. M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241–245 (2006).
Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540–551 (2015).
Qiling, X. in Molecular Methods in Developmental Biology Vol. 127 (ed. Guille, M.) 125–132 (Humana Press, Totowa, NJ, 1999).
Kelsh, R. N. et al. Zebrafish pigmentation mutations and the processes of neural crest development. Development 123, 369–389 (1996).
Inaba, M., Yamanaka, H. & Kondo, S. Pigment pattern formation by contact-dependent depolarization. Science 335, 677 (2012).
Ma, X. et al. Polo-like kinase 1 coordinates biosynthesis during cell cycle progression by directly activating pentose phosphate pathway. Nat. Commun. 8, 1506 (2017).
Noda, T. & Amano, F. Differences in nitric oxide synthase activity in a macrophage-like cell line, RAW264.7 cells, treated with lipopolysaccharide (LPS) in the presence or absence of interferon-gamma (IFN-gamma): possible heterogeneity of iNOS activity. J. Biochem. 121, 38–46 (1997).
Lohman, J. R. & Remington, S. J. Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments. Biochemistry 47, 8678–8688 (2008).
Fan, Y. & Ai, H. W. Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments. Anal. Bioanal. Chem. 408, 2901–2911 (2016).
Ostergaard, H., Henriksen, A., Hansen, F. G. & Winther, J. R. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J. 20, 5853–5862 (2001).
Markvicheva, K. N. et al. A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg. Med. Chem. 19, 1079–1084 (2011).
Bilan, D. S. et al. HyPer-3: a genetically encoded H2O2 probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem. Biol. 8, 535–542 (2013).
Williamson, D. H., Lund, P. & Krebs, H. A. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103, 514–527 (1967).
We thank S.J. Remington for the roGFP1 vector; V.V. Belousov for the HyPer and HyPerRed vectors; J. Du for the pTol2 vector; J. Yi for the psPAX2 and pMD2.G vectors; N. Su, L. Huang, Q. Wang, P. Ni, and H. Zi for technical assistance; and S.C. Tribuna for secretarial assistance. This research was supported by the National Key Research and Development Program of China (2017YFC0906900, 2017YFA050400, 2016YFA0100602, and 2017YFA0103302), the NSFC (31722033, 91649123, 31671484, 31225008, 31470833, 91749203, 81525010, and 81420108017), the Shanghai Science and Technology Commission (14XD1401400, 16430723100, and 15YF1402600), the Young Elite Scientists Sponsorship Program by Cast, Shanghai Young Top-notch Talent, the State Key Laboratory of Bioreactor Engineering, the Fundamental Research Funds for the Central Universities, the US National Institutes of Health (HL061795, HG007690, and GM107618 to J.L.), and the American Heart Association (D700382 to J.L.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
1. Tao, R. et al. Nat. Methods 14, 720–728 (2017): https://doi.org/10.1038/nmeth.4306
2. Zhao, Y. et al. Cell Metab. 21, 777–789 (2015): https://doi.org/10.1016/j.cmet.2015.04.009
3. Zhao, Y. et al. Nat. Protoc. 11, 1345–1359 (2016): https://doi.org/10.1038/nprot.2016.074
Integrated supplementary information
(a and b) Fluorescence imaging (top) and fluorescence changes (bottom, n = 6 cells) in HeLa cells simultaneously expressing cytosol-localized iNapc and cytosol-localized pHRFP in response to 200 µM diamide (a) or 2 mM oxamate (b). (c and d) Fluorescence imaging (top) and fluorescence changes (bottom, n = 6 cells) in HeLa cells simultaneously expressing mitochondria-localized iNapc, nuclear-localized iNapc, and mitochondrial-localized pHRFP in response to 200 µM diamide (c) or 2 mM oxamate (d). Data are the mean ± s.d. All p values were obtained using unpaired two-tailed Student’s t test. *p < 0.05, ***p < 0.001. Scale bars, 10 µm.
(a and b) Fluorescence images (a) and quantification (b) of pH dynamics during cell division. Scale bars, 10 µm.
(a) Sample gating strategy for forward and side scatter (FSC/SSC). (b and c) Sample gating strategy for negative cells (b) and positive cells (sensor-expressing cells, c).
Supplementary Figure 4 Flow cytometry analysis of the pH state in resting and activated mouse macrophages.
(a) Cytosolic or mitochondrial pH detection in resting or activated RAW264.7 mouse macrophages by flow cytometry. (b) Quantitative data for cytosolic or mitochondrial pH sensor fluorescence were obtained from three or more independent detections by flow cytometry. Data are the mean ± s.e.m. All p values were obtained using unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.
(a and b) In vivo fluorescence imaging of zebrafish larvae expressing iNapc in response to 50 mM H2O2 (a) or 5 µM rotenone (b).
About this article
Cite this article
Zou, Y., Wang, A., Shi, M. et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors. Nat Protoc 13, 2362–2386 (2018). https://doi.org/10.1038/s41596-018-0042-5
Inflammation, epigenetics, and metabolism converge to cell senescence and ageing: the regulation and intervention
Signal Transduction and Targeted Therapy (2021)
Nature Communications (2021)
Signal Transduction and Targeted Therapy (2020)
Protein & Cell (2019)