Journal
NATURE PROTOCOLS
Volume 11, Issue 7, Pages 1219-1228Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.062
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Funding
- National Creative Research Initiative programs of the National Research Foundation of the Ministry of Science, ICT and Future Planning of Korea [2012R1A3A2048814, 2015R1A3A2033475]
- Basic Science Research Program [NRF 2013R1A1A2013219]
- Korea Mouse Phenotyping Project [2013M3A9D5072550]
- National Natural Science Foundation of China [21376117]
- Jiangsu Natural Science Funds for Distinguished Young Scholars [BK20140043]
- National Research Foundation of Korea [2015R1A3A2033475] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROSROSROS) such as hypochlorous acid (HOCOCl) to kill the invading pathogens. However, excess amounts of HOCOCl induce oxidative damage of functional biomolecules such as DNANA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCOCl probe, as well as applications thereof, with which to detect HOCOCl 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 HOCOCl, 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 HOCOCl over other ROSROSROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCOCl 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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