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 death^1,2. Oxidative stress contributes to aging and to the development of many life-threatening diseases including cancer, diabetes, cardiovascul....

Protocol

1. Preparation of Reagents

1. Preparation of media for C. elegans growth (in this case, wild-type animals and flcn-1(ok975) mutant animals).
   1. Prepare Modified Youngren's Only Bacto-peptone (MYOB) dry mix containing 5.5 g of Tris HCl, 2.4 g of Tris base, 31 g of Bactopetone, 20 g of NaCl and 0.08 g of cholesterol. Mix well with shaking.
      NOTE: This mix is sufficient to prepare 10 L of MYOB medium.
      NOTE: Normal growth medium (NGM) plates could be used instead of MYOB plates²⁷. However, the same type of plates should be used for valid comparisons between strains and between independent repeats. In this p....

Results

Comparing wild-type C. elegans to flcn-1(ok975) mutant animals

Here we used 100 mM PQ to determine the resistance of wild-type C. elegans animals compared to flcn-1(ok975) which has been shown to resist oxidative stress, heat, and anoxia²³. After 4 hr of treatment, 48.3% of wild-type survived as compared to 77.8% survival in flcn-1(ok975) animals. As expected, flcn-1(ok975) mutant animals were more resistant to 100 mM PQ compared.......

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 juglone^25,26,29-32. Here we describe a protocol that measures oxid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 60 x15 mm	Fisher	FB0875713A
Pipet 10 ml	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