Abstract
Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPDSA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPDSA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N,N-dimethyl formamide at 85 °C to form cyanine-piperazine (CP). The second step is the sulfonylation of CP with dansyl chloride in anhydrous dichloromethane. CPDSA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow–derived neutrophils (BMDNs). The preparation of probe CPDSA takes 2 d, and experiments in cells and mice take 12–13 d.
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Acknowledgements
This research was supported by a grant from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean government (grant no. 2012R1A3A2048814), by the Basic Science Research Program through the NRF funded by the Ministry of Education (grant no. 2013R1A1A2008511), and by the Yonsei University College of Medicine (grant no. 6-2014-0135 to J.-H.R.) and the National Natural Science Foundation of China (grant no. 21402057).
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J. Yin, Y.K. and G.K. conducted experiments; D.K., D.L. and Y.H. measured partial data; and J. Yin, Y.K., G.K., J.-H.R. and J. Yoon designed experiments and wrote the paper.
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Supplementary Figure 1 Fluorescent titration of probe CPDSA.
Fluorescent titration of probe CPDSA (10 uM) upon addition of GSH in HEPES (10 mM, pH = 7.4) containing 10% DMSO. Each spectrum was recorded at 20 min after the addition of GSH. (λex = 730 nm, λem = 736 nm, slit: 10/10 nm)
Supplementary Figure 2 Confocal microscope images following addition of probe CPDSA to HeLa cells.
Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). Cell images were obtained using an excitation wavelength of 635 nm and a band‐path (655−755 nm) emission filter. (A) Fluorescence image of HeLa cells; (B) Fluorescence image of HeLa cells incubated with probe CPDSA (20 μM) for 20 min; (C) Fluorescence image of HeLa cells pretreated with N-methylmaleimide (NMM, 1 mM) for 20 min and incubated with probe CPDSA (20 μM) for 20 min; (D) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with cysteine (100 μM) and incubated with probe CPDSA (20 μM) for 20 min, (E) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with homocysteine (100 μM) and incubated with probe CPDSA (20 μM) for 20 min; (F) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min, and then added with GSH-MEE (100 μM) and incubated with probe CPDSA (20 μM) for 20 min. Scale bar: 15 μm.
Supplementary Figure 3 The time-dependent confocal microscope images following addition of probe CPDSA to HeLa cells.
Confocal microscope images following addition of probe CPDSA to HeLa cells (The data is collected from FV1200, Olympus, Japan). HeLa cells were incubated with 20 μM probe CPDSA for 20 min and washed with DPBS and exchanged with new media. The time-dependent fluorescence images of probe CPDSA were acquired by confocal microscopy. Cell images were obtained using an excitation wavelength of 635 nm and a band-path (655−755 nm) emission filter.
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Yin, J., Kwon, Y., Kim, D. et al. Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice. Nat Protoc 10, 1742–1754 (2015). https://doi.org/10.1038/nprot.2015.109
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DOI: https://doi.org/10.1038/nprot.2015.109
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