Published: Vol 8, Iss 6, Mar 20, 2018 DOI: 10.21769/BioProtoc.2774 Views: 15317
Reviewed by: Neelanjan BoseMichael EnosAnonymous reviewer(s)
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Abstract
Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS levels photometrically in Caenorhabditis elegans using 2’,7’-dichlorofluorescein diacetate (H2DCFDA), a cell permeant tracer.
Keywords: Reactive oxygen speciesBackground
In situ detection of intracellular reactive oxygen species (ROS) levels in the living organism using fluorescent probe 2’,7’-dichlorofluorescein diacetate (H2DCFDA) has been broadly performed by those researchers who work in the field of oxidative stress and related diseases. The non-polar and non-ionic probe, H2DCFDA, can easily penetrate the cellular membrane and is enzymatically deacetylated by esterases. This biochemical reaction turns H2DCFDA into the non-fluorescent compound H2DCF which is then rapidly oxidized to highly fluorescent 2’,7’-dichlorofluorescein (DCF) in the presence of ROS (Figure 1A). Therefore, fluorescence signals from H2DCFDA probe demonstrate important information for the quantification of ROS at single cell level (Labuschagne and Brenkman, 2013). The Caenorhabditis elegans model system provides an excellent in vivo experimental environment for evaluating molecular mechanisms of ROS pathophysiology due to their short lifespan, simplicity, and ease of genetic manipulation (Labuschagne and Brenkman, 2013; Miranda-Vizuete and Veal, 2017; Yoon et al., 2017) (Figure 1B). Here, we describe a simple protocol to measure the levels of time-course ROS generation in C. elegans using H2DCFDA under normal and heat- or chemically-induced oxidative stress conditions. Using this protocol, we determined the effects of H2DCFDA concentration and number of tested worms on DCF fluorescence signal (Figure 2).
Figure 1. ROS detection. A. Production of florescent DCF by intracellular ROS. B. Measurement of intracellular ROS using molecular probe (H2DCFDA) in C. elegans.
Figure 2. Change in DCF fluorescence signals depends on experimental conditions. A. The expression of gcs-1(promoter)::GFP (oxidative stress marker) transgene by oxidative stress. Heat (30 °C for 2 h) induced the expression of gcs-1(promoter)::GFP transgene in the intestines of live nematodes. B. Concentration-response curves were plotted in terms of the mean value of DCF fluorescence signals induced by 12.5, 25, 50, and 100 μM of H2DCFDA using 50 nematodes for each experiment. C. Changes in DCF fluorescence signals depending on the number of tested nematodes (10, 20, 50, and 100) were assessed using 25 μM of H2DCFDA.
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Acknowledgments
This work was supported in part by the National Research Foundation of South Korea (NRF-2017R1C1B5015695) to DSC, Brody Brothers Endowment Grant (21602-664261), NIH (1R15GM112174-01A1), NSF (MCB1714264) to M-H.L. The Caenorhabditis Genetics Center (CGC) is supported by the National Institutes of Health – Office of Research Infrastructure Programs (P40 OD010440). This protocol has been adapted from Yoon et al., 2017. The authors have declared that no competing interests exist.
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Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Yoon, D. S., Lee, M. and Cha, D. S. (2018). Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate. Bio-protocol 8(6): e2774. DOI: 10.21769/BioProtoc.2774.
Category
Developmental Biology > Cell signaling > Stress response
Biochemistry > Other compound > Oxygen
Cell Biology > Cell signaling > Intracellular Signaling
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