Published: Vol 7, Iss 17, Sep 5, 2017 DOI: 10.21769/BioProtoc.2545 Views: 24623
Reviewed by: Dennis NürnbergPooja SaxenaAnonymous reviewer(s)
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Abstract
Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ROS by using an oxidant-sensing probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in cyanobacteria. This method provides reliable, simple, rapid and cost effective means for detection of ROS in cyanobacteria.
Keywords: Reactive oxygen speciesBackground
Cyanobacteria are the most ancient oxygenic photoautotrophs; they play an important role in the biomass production in both aquatic and terrestrial ecosystems and serve as source of various value-added products (Vaishampayan et al., 2001; Häder et al., 2007; Fischer, 2008). In recent years the depletion of the ozone layer has resulted in an increase in solar ultraviolet radiation (UVR) influx, which is harmful to all organisms residing on Earth including cyanobacteria (Holzinger and Lutz, 2006). The UVR harms cyanobacteria directly by acting on DNA/proteins or indirectly through oxidative damage from reactive oxygen species (ROS) (He and Häder, 2002). In plants, algal and mammalian cells various fluorescence and chemiluminescence methods have been used for detecting ROS (Crow, 1997; He and Häder, 2002; Soh, 2006; Wu et al., 2007; Palomero et al., 2008).
2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) is a non-fluorescent, cell-permeable dye which is hydrolyzed intracellularly into its polar, but non-fluorescent form DCFH on the action of cellular esterases and thus is retained in the cell. Oxidation of DCFH by the action of intracellular ROS and other peroxides turns the molecule into its highly fluorescent form 2’,7’-dichlorofluorescein (DCF) that can be detected by various fluorescent methods (He and Häder, 2002; Rastogi et al., 2010; Singh et al., 2014) (Figure 1). Although DCFH-DA is widely used for the detection of ROS, it should be noted, however, that the dye cannot be used as an indicator for a specific form of ROS (Marchesi et al., 1999).
Figure 1. Mechanism of action of DCFH-DA probe inside the cell (Adapted from He and Häder, 2002)
Materials and Reagents
Equipment
Software
Procedure
A flow chart of the sample preparation is shown in Figure 2.
Figure 2. Flow chart showing various steps involved in the protocol
Data analysis
The UVR irradiated cells were analyzed using a Nikon Eclipse Ni fluorescence microscope processed by NIS-Elements (BR) imaging software. The microscope was equipped with the following filter set: UV: (DAPI) EX 340 nm EM 488 nm, blue: (FITC) EX 495 nm EM 510 nm and green: (PI 550) EX 550 nm EM 650 nm. Cells were imaged in the epifluorescence mode with a 20x objective lens. The image analysis was performed by NIS-Elements (BR) imaging software provided by Nikon and images were saved in JPEG format. In addition, the fluorescence of the samples was measured by a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) with an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. The data of the fluorescence spectra were exported to excel and the fluorescence intensity values at 525 nm were extracted. A bar diagram was plotted with SigmaPlot 11 software. All fluorescence measurements were performed at room temperature. All results are presented as mean values of three replicates for fluorescence spectrophotometer analysis and random sites of filaments were used for fluorescence microscopy. All data were analysed by one-way analysis of variance (Brown, 2005). Once a significant difference was detected post hoc multiple comparisons were made by using the Tukey test. The level of significance was set at 0.05 for all tests. All statistical analyses were performed by using SigmaPlot 11 software.
Notes
Recipes
Acknowledgments
Rajneesh and Jainendra Pathak are thankful to the Department of Biotechnology (DBT-JRF/13/AL/143/2158) and the Council of Scientific and Industrial Research (09/013/0515/2013-EMR-I), New Delhi, India, respectively, for the financial support in the form of fellowships. SP Singh acknowledges the DST-SERB and UGC for Early Career Research Award and UGC Start-Up Research Grant, respectively. We are also thankful to the Interdisciplinary School of Life Sciences (ISLS), BHU, Varanasi, India, for providing access to the fluorescence microscopy facility. This protocol was adapted from procedures published by Rastogi et al., 2010.
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Rajneesh, , Pathak, J., Chatterjee, A., Singh, S. P. and Sinha, R. P. (2017). Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA). Bio-protocol 7(17): e2545. DOI: 10.21769/BioProtoc.2545.
Category
Microbiology > Microbial biochemistry > Other compound
Biochemistry > Other compound > Reactive oxygen species
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