Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates
Summary
C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.
Abstract
Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.
Introduction
In eukaryotes, oxidative phosphorylation taking place in the electron transport chain of the mitochondria is the main driver of energy production in the form of ATP. Reactive oxygen species (ROS) are a natural byproduct of this process. Despite their important role as signaling molecules, excessive ROS can lead to DNA damage, protein carbonylation, and lipid oxidation. An imbalance between ROS production and detoxification causes oxidative stress, which leads to energy depletion, cellular damage, and triggers cell death1,2. Oxidative stress contributes to aging and to the development of many life-threatening diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases3-9.
Cells have evolved enzymatic and non-enzymatic defense strategies to maintain proper ROS levels and to protect their constituents against oxidative damage1,2. Superoxide dismutase (SOD) enzymes act first to convert superoxide to H2O2, which is later converted to water by catalase or peroxidase enzymes. Non enzymatic defense strategies include mostly molecules that react faster with ROS as compared to cellular macromolecules, protecting essential cellular components. Despite the protective role of ROS detoxifying enzymes, some ROS molecules escape the antioxidant defense mechanisms and lead to oxidative damage. Detection, repair, and degradation of the damaged cellular components are essential defense strategies during oxidative stress1,2.
Signaling pathways involved in stress resistance and specifically oxidative stress are highly evolutionarily conserved10,11. Unlike cell culture experiments where organismal conditions are only partially reproduced, the study of oxidative stress in model organisms12,13 has great significance. C. elegans is a free-living nematode that can be easily and inexpensively cultured on a bacterial lawn on agar media. It is small in size (about 1 mm in length) and normally grows as a self-fertilizing hermaphrodite, which facilitates genetic manipulations. It has a rapid life cycle and a high reproductive capacity, producing about 300 offspring per generation, making it a powerful tool to perform large-scale genetic screens14. The C. elegans genome is fully sequenced and 40-50% of the genes are predicted to be homologues of human disease-associated genes15-18. The knockdown of genes of interest using RNAi is rapid and easy in C. elegans. Gene down regulation could be achieved by feeding animals the E. coli bacteria that harbor a plasmid expressing the double-stranded RNA that targets the mRNA of interest19. Therefore, determination of gene function using large scale RNAi screens has great impact on understanding human diseases including cancer 20,21.
Studies of oxidative stress resistance in C. elegans have led to the identification of conserved mechanisms of resistance to oxidative stress13,22. Some pathways identified are common pathways that modulate longevity and resistance to other stresses as well such as hypoxia, heat, and osmotic stress. These pathways include the insulin signaling, TOR signaling, and autophagy. Other key pathways involve detoxification of ROS such as superoxide dismutase enzymes and catalase enzymes, or in damage repair such as heat shock and chaperone proteins11,13,22.
This protocol describes how to determine the resistance to oxidative stress of C. elegans in liquid. We used flcn-1(ok975) and wild-type animals to demonstrate the protocol since we have previously shown an increased resistance to oxidative stress upon loss of flcn-1(ok975) in C. elegans23. We have also shown that this increased resistance depends on AMPK and autophagy, a signaling axis that improves cellular bioenergetics and promotes stress resistance 23. PQ is an oxidative stressor that interferes with the electron transport chain to produce reactive oxygen species24. The same assay could be adapted and other ROS sources or ROS generating compounds could be used such as H2O2 and rotenone. Similar assays have been developed on plates where low concentrations of PQ are used25,26. The advantage of this assay is that it is very fast, and the results could be obtained in one day. Additionally, the total volume of liquid used to perform the oxidative stress resistance assay in 96 well plates is low as compared to the volume used to prepare PQ-containing plates. Therefore, the amount of PQ used is in the liquid assay is low, which renders the assay inexpensive and limits the production of toxic wastes. However, limitations of this assay as compared to plate assays include the lack of food in the liquid assay and the lower concentration of oxygen in liquid as compared to air. These are important factors that in some cases, might influence the results. Therefore, confirming reproducibility using other methods of oxidative stress resistance is recommended to support results obtained in this assay.
Protocol
Representative Results
Discussion
C. elegans is an attractive model organism to study genetically oxidative stress resistance in vivo since it can be easily cultured, and rapidly leads to a large number of genetically identical offspring. Multiple methods to measure oxidative stress resistance have been previously described and they are based on the supplementation of culture plates with various ROS sources such as PQ, rotenone, H2O2, and juglone25,26,29-32. Here we describe a protocol that measures oxid…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge the Caenorhabditis Genetics Center for C. elegans strains. Funding support was provided by the Terry Fox Research Institute. We also acknowledge support granted to E.P. from the Rolande and Marcel Gosselin Graduate Studentship and the CIHR/FRSQ training grant in cancer research FRN53888 of the McGill Integrated Cancer Research Training Program.
Materials
| Agar bacteriological grade | Multicell | 800-010-LG | |
| Bacteriological peptone | Oxoid | LP0037 | |
| Sodium chloride biotechnology grade | Bioshop | 7647-14-5 | |
| Cholesterol | Sigma | C8503-25G | |
| UltraPure tris hydrochloride | Invitrogen | 15506-017 | |
| Tris aminomethane | Bio Basic Canada Inc | 77-86-1 | |
| IPTG | Santa Cruz Biotechnology | sc-202185A | |
| Ampicillin | Bioshop | 69-52-3 | |
| Yeast extract | Bio Basic Inc. | 8013-01-2 | |
| Methyl viologen dichloride hydrate | Aldrich chemistry | 856177-1G | |
| Petri dish 60x15mm | Fisher | FB0875713A | |
| Pipet 10ml | Fisher | 1367520 | |
| Potassium phosphate monobasic | G-Biosciences | RC-084 | |
| Magnesium sulfate heptahydrate | Sigma | M-5921 | |
| Sodium phosphate dibasic | Bioshop | 7558-79-4 | |
| Discovery v8 stereo zeiss microscope | |||
| 96 well clear microtiter plate | |||
| flcn-1 RNAi source | Ahringer Library | ||
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