Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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, cardiovascular and neurodegenerative diseases^3-9.

Cells have evolved enzymatic and non-enzymatic defense strategies to maintain proper ROS levels and to protect their constituents against oxidative damage^1,2. 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 stress^1,2.

Signaling pathways involved in stress resistance and specifically oxidative stress are highly evolutionarily conserved^10,11. Unlike cell culture experiments where organismal conditions are only partially reproduced, the study of oxidative stress in model organisms^12,13 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 genes^15-18. 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 stress^13,22. 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 proteins^11,13,22.

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 used^25,26. 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.

Protokół

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 protocol, we only used MYOB plates.
   2. Prepare 1 L of MYOB medium by adding 6 g of MYOB dry mix, 17 g of agar and make up to 1 L with H₂O and autoclave for 45 min at 122 °C. Pour plates and let dry at RT for 2 days.
   3. Using sterile technique, inoculate 100 ml of culture of LB broth medium with E. coli OP50 bacteria and grow O/N at 37 °C.
   4. Seed MYOB plates with E. coli OP50 bacteria. Allow it to grow at RT for 48 hr.
2. Preparation for gene downregulation using RNAi
   1. Prepare MYOB RNAi-feeding plates. Proceed as in steps 1.1.1 and 1.1.2. After autoclaving, allow the medium to cool down to about 55 °C and add 1 ml of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) and 1 ml of 100 mM ampicillin.
   2. Streak E. coli HT115 bacteria transformed with a plasmid, which expresses either control EV or flcn-1 specific RNAi on Ampicillin (50 µg/ml)/tetracyclin (15 µg/ml) agar plates. Grow culture O/N at 37 °C.
   3. Pick colonies and inoculate E. coli HT115 bacteria transformed with a plasmid which expresses either control EV or flcn-1 specific RNAi and grow culture in LB/Ampicillin (50 µg/ml) medium and O/N at 37 °C.
   4. Seed plates with E. coli HT115 EV bacteria or HT115 flcn-1 RNAi bacteria.
      NOTE: Keep plates 48 hr at RT before usage. If RNAi knockdown by feeding is in effective, use other methods of delivery of the ds-RNA ²⁸.
3. Inducing oxidative stress using paraquat (PQ):
   1. Prepare M9 buffer by mixing 3 g of KH₂PO₄, 6 g of Na₂HPO₄, 5 g of NaCl and 0.25 g of MgSO₄·7 H₂O. Bring up to 1 L with distilled water. Autoclave solution for 45 min at 122 °C and store bottles at RT.
   2. On the day of the assay, prepare the M9/PQ-containing solution. For this protocol, use 100 mM PQ (0.1028 g of PQ dissolved in 4 ml of M9 buffer fills an entire 96 well plates with 40 µl of M9/PQ per well).
      CAUTION: PQ is a hazardous and highly toxic compound. Handle according to appropriate guidelines.
      NOTE: When first running a new strain or a new condition, it is recommended to assay survival in increasing concentrations of PQ to determine the best concentration to use in order to kill the animals within 7-10 hr.

2. Preparation of Age-synchronized L4 Population

1. Transfer 20 gravid adult hermaphrodites to a fresh MYOB plate seeded with E. coli OP50 bacteria. Prepare several plates based on the number of worms needed during the assay.
2. Allow them to lay eggs for 6 hr and then using a platinum wire worm pick remove the mothers. Check the plates under the microscope to assess the number of eggs laid.
3. Allow the eggs to hatch and grow at 20 °C for 48 hr. At this time, the animals are in a late L4/young adult stage. Pick same stage animals and transfer to the PQ solution-containing wells.
   NOTE: Since 48 hr incubation only applies to mutants that grow similarly to wild-type animals, it is important to monitor developmental rate of newly tested mutants to ensure it conforms to the wild-type development rate, otherwise, other timelines should be used.
4. Alternatively, use synchronization techniques. Generate synchronized population of L1 larvae worms using hypochlorite solution (25 ml water, 125 ml of 5.25% sodium hypochlorite, 50 ml of 4 M NaOH; wrap bottle with aluminum foil to isolate from light). However, for this assay, it is not recommended, since intense bleaching might affect the behavior of the animals during the assay.
   CAUTION: Handle hypochlorite solution according to guidelines.
   NOTE: When using RNAi to down regulate the gene of interest, synchronize the hermaphrodites as described in section 2, with the exception of the transfer of the mothers to RNAi plates seeded with the specific RNAi bacteria. When the knockdown with the RNAi is weak, feeding for multiple generations is recommended.

3. Performing the Oxidative Stress Resistance Assay

1. Pipette 40 µl of the 100 mM PQ solution (prepared fresh) to every well of a 96 well plate. Use at least 12 wells as replicates of every condition (treatment, mutant, etc.). Using a platinum wire worm pick, transfer 5-8 L4 larvae animals to every well indicating the start time and the end time.
2. When starting another condition, note the start time and the end time. Place the plate at 20 °C in the incubation time between transferring the worms and scoring dead animals an hour later from the start time.
3. Using the dissecting microscope, count survival every hr. Gently shake the plate before starting to count. Indicate worms that do not move, even after spotting a high-intensity light, as dead animals. Also indicate the number of animals alive.
   NOTE: Looking at worm shape, tails and head movement at high magnification, help determine dead worms from alive.
4. Repeat this step every hour until most worms are dead.

4. Determination of Survival Percentage at Every Time Point

1. Calculate the total number of animals per condition by adding the total number of animals transferred to every well. Ignore the animals that are damaged or killed upon transfer.
2. For every time point, calculate the total number of dead animals per condition (sum of dead animals in the 12 wells). For every time point, calculate percentage of death (number of dead animals divided by total number of animals and multiplied by 100) and percentage of survival (100 minus percent death).
3. Repeat this assay at least 3 independent times.

Wyniki

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...

Dyskusje

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...

Materiały

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 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