Abstract
The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used MitoSOX and CellROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses.
Keywords: Zebrafish, Reactive Oxygen Species, Oxidative stress
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 2. Representative analysis of oxidative stress in WT and mutant embryos using MitoSOX and CellROX staining. A-C. Representative quantitative analysis by flow cytometry of oxidative stress using MitoSOX Red (A and B) or CellROX Green (C) on 4 and 5 dpf mutant and control embryos. Error bars indicate standard deviation; **P < 0.01; ***P < 0.001 using an unpaired Student’s t-test. D. Whole mount staining with the MitoSOX or CellROX probes followed by confocal microscopy in the tail region of WT and mutant embryos at the indicated stages. As internal positive control, embryos at the stages of 4 or 5 dpf were subjected to tail resection 30 min before being stained with MitoSOX or CellROX solutions. If tail resection cannot be performed, because of the stage analyzed or because it could interfere with the specific conditions of the experiment or the fish line used, an alternative positive control could be represented by the treatment of the embryos as indicated in Mugoni et al. (2014). Yellow arrowheads in the insets indicate MitoSOX- or CellROX-positive cells in CHT regions. CHT: Caudal Hematopoietic Tissue (from Rissone et al., 2015).
Recipes
Acknowledgments
This protocol was modified from Behra et al. (2012) and Mugoni et al. (2014), and developed in the Dr. Fabio Candotti Lab. Disorders of Immunity Section, Genetics and Molecular Biology Branch, NHGRI, NIH. This protocol was supported by funding from the Intramural Research Program of the National Human Genome Research Institute (to F. Candotti, R. Sood, and S. Chandrasekharappa).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Dear Bio-protocol user,Thank you very much for your question. As stated in the title, our protocol is intended for the detection of ROS in Zebrafish using two specific reagents: the MitoSOX and the CellROX. While the MitoSOX is supposed to be more specific for superoxide generated in Mitochondria, CellROX is a more generic probe, labeling any kind of ROS in all the cellular districts. However, following our protocol, you should be able to use your favorite probe in order to label specific kind of ROS. In any case, as mentioned in the protocol (“…the length of the incubation should be optimized based on the developmental stage, the staining solution and the oxidative status of the sample”), each researcher must find the optimal experimental conditions for the samples that will be analyzed. It’s impossible to prepare a protocol detailed enough to take in consideration all the possible variants (fish line, developmental stage, specific tissue/organ or cell type, oxidative status, probe concentration, ROS sub-type, etc…). Concerning the information about the Flow Cytometry approach, there is no specific knowledge that a FACS/sorter technician need to know to analyze the samples. Again, we omitted the info about that (with the exception of the instrument we used), basically because the conditions must be empirically determined and set by the researcher. Your specific conditions could vary depending for example on the zebrafish line in use, the cell type, the level of cell viability, the flow cytometer instrument in use, the power of the lasers, the threshold that you want to use, etc…I'm sorry I can't be more helpful. If you have more specific questions, do not hesitate to contact me again and I will try to do my best to help you. Best regards,Alberto Rissone