Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate

Background

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.