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Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms

Nature Protocols volume 11, pages 1219–1228 (2016)Cite this article

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Abstract

During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROS) such as hypochlorous acid (HOCl) to kill the invading pathogens. However, excess amounts of HOCl induce oxidative damage of functional biomolecules such as DNA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCl probe, as well as applications thereof, with which to detect HOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCl over other ROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2–3 d, after which it can be used for up to 5 h in the bioimaging of living cells.

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Figure 1
Figure 2: The selectivity of R19S toward various ROS/RNS and the concentration-dependent responses to HOCl.
Figure 3: Comparisons of selectivity to HOCl and pH-dependent responses among R19S, APF and DHR.
Figure 4: Imaging of DUOX-dependent HOCl production in the Drosophila intestine following bacterial infection.
Figure 5: Confocal microscopy images of GFP-tagged PAO1 and R19S in mouse bone-marrow-derived neutrophils.
Figure 6: Flow cytometry analysis of R19S in neutrophils from BAL fluids in a mouse model of lung infection.

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Acknowledgements

This work was supported by grants from the National Creative Research Initiative programs of the National Research Foundation of the Ministry of Science, ICT and Future Planning of Korea (2012R1A3A2048814 to J.Y. and 2015R1A3A2033475 to W.-J.L.). K.-A.L. is supported by the Basic Science Research Program (NRF 2013R1A1A2013219), and J.-H.R. is supported by the Korea Mouse Phenotyping Project (2013M3A9D5072550). X.C. acknowledges support from the National Natural Science Foundation of China (21376117) and the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043). We thank E.J. Park (National Cancer Center, Korea) for critical help in MPO mouse work.

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Author notes

  1. Xiaoqiang Chen and Kyung-Ah Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Nano Science, Ewha Womans University, Seoul, South Korea

    Xiaoqiang Chen, Gyungmi Kim & Juyoung Yoon

  2. Department of Bioinspired Science (WCU), Ewha Womans University, Seoul, South Korea

    Xiaoqiang Chen, Gyungmi Kim & Juyoung Yoon

  3. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing, People's Republic of China

    Xiaoqiang Chen & Xintong Ren

  4. National Creative Research Initiative Center for Hologenomics, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea

    Kyung-Ah Lee & Won-Jae Lee

  5. School of Biological Science, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea

    Kyung-Ah Lee & Won-Jae Lee

  6. Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul, South Korea

    Jae-Chan Ryu & Ji-Hwan Ryu

  7. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea

    Jae-Chan Ryu & Ji-Hwan Ryu

  8. BK 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea

    Jae-Chan Ryu & Ji-Hwan Ryu

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Contributions

J.Y., W.-J.L. and J.-H.R. designed the experiments; X.C., X.R. and G.K. performed the chemical synthesis and fluorescence assays in vitro; K.-A.L., J.-C.R. and J.-H.R. performed the biological experiments; J.Y., W.-J.L., J.-H.R. and X.C. wrote the paper.

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Correspondence to Ji-Hwan Ryu, Won-Jae Lee or Juyoung Yoon.

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Chen, X., Lee, KA., Ren, X. et al. Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms. Nat Protoc 11, 1219–1228 (2016). https://doi.org/10.1038/nprot.2016.062

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