Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays

Materials and Reagents

1. Petri dishes 60 x 15 mm 500/cs (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875713A )
   
2. Petri dishes 35 x 10 mm 500/cs (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875711YZ )
   
3. Autoclave tape
   
4. Aluminum foil
   
5. 99.95% Platinum, 0.05% Iridium Wire (3 ft/pk) (Tritech Research, catalog number: PT-9901 )
   
6. OP50 E. Coli bacteria (University of Minnesota, C. elegans Genetics Center, N/A)
   
7. Experimental and control C. elegans strains (University of Minnesota, C. elegans Genetics Center, N/A)
   
8. Eggs from experimental and control C. elegans strains
   
9. Methyl viologen dichloride hydrate (paraquat) (Sigma-Aldrich, catalog number: 856177 )
   
10. FUdR (5-fluoro-2’-deoxyuridine) (Sigma-Aldrich, catalog number: F0503 )
    
11. 5-hydroxy-1,4-naphthoquinone (juglone) (Sigma-Aldrich, catalog number: H47003 )
    
12. 100% ethanol
    
13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
    
14. Bacto peptone (BD, catalog number: 211677 )
    
15. Agar (Sigma-Aldrich, catalog number: A1296 )
    
16. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
    
17. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: M1880 )
    
18. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C3881 )
    
19. Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P2222 )
    
20. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
    
21. Tryptone (Sigma-Aldrich, catalog number: T7293 )
    
22. Yeast extract (Sigma-Aldrich, catalog number: 70161 )
    
23. 0.5% cholesterol (see Recipes)
    
24. 1 M MgSO₄ (see Recipes)
    
25. 1 M CaCl₂ (see Recipes)
    
26. KPI (see Recipes)
    
27. Nematode growth medium (NGM) (see Recipes)
    
28. 1 M paraquat (see Recipes)
    
29. 3.33 M paraquat (see Recipes)
    
30. 0.1 M FUdR stock solution (see Recipes)
    
31. Juglone stock solution (12 mM) (see Recipes)
    
32. 2 YT medium (see Recipes)
    

Equipment

1. Erlenmeyer flask (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB5006000 )
   
2. M50 stereomicroscope (Leica, model: 10450154 )
   
3. Pipetor (Gilson, catalog number: F167300 )
   
4. Autoclave (Tuttnauer, model: 6690 )
   
5. Stirring hotplate (Corning, catalog number: 6795-620 )
   
6. Centrifuge (Eppendorf, model: 5430 )
   
7. Refrigerated incubator (Thermo Fisher Scientific, Thermo scientific^TM, model: 51028064 ; 37-20)
   
8. Bunsen burner (Humbolt, catalog number: H5870 )
   

Software

1. GraphPad Prism software (we use Version 5.01)

Procedure

1. Paraquat development assay - sensitivity to chronic oxidative stress during development
   1. Prepare NGM media (see Recipes) in Erlenmeyer flask (Note 1).
      
   2. Cover the top of Erlenmeyer flask with aluminum foil and use autoclave tape to secure it in place.
      
   3. Autoclave NGM media and MgSO₄, CaCl₂ and KPI solutions (see Note 2).
      
   4. Cool media to ~55 °C (see Note 3) while stirring.
      
   5. Add MgSO₄, CaCl₂ and KPI (according to Recipes) while stirring.
      
   6. Pour control plates (no paraquat): 10 ml of media in 60 mm Petri dish (see Notes 4 and 5).
      
   7. Add paraquat (methyl viologen) (according to Recipes) while stirring (see Note 6). Allow ~60 sec for paraquat to be mixed into the media.
      
   8. Pour plates: 10 ml of media in 60 mm Petri dish.
      
   9. Repeat steps A7 and A8 for each concentration of paraquat (see Note 7).
      
   10. Allow plates to solidify and dry overnight at room temperature.
       
   11. Seed plates with 100 µl of OP50 bacteria overnight culture.
       
   12. Allow bacteria to dry and grow at room temperature for 2 days.
       
   13. Transfer equal numbers of eggs from each strain being tested to control plates and plates containing paraquat (Note 8) ensuring that you don’t transfer any eggs that have hatched into the first larval stage of development (L1 worms).
       
   14. Check to ensure that eggs have hatched the following day.
       
   15. Monitor the developmental stage of the worms daily (see Note 9).
       
   16. Expected results: Wild-type N2 worms should develop to fertile adults on plates up to about 0.35 mM paraquat.
       
   17. Positive controls: clk-1, sod-2 both arrest at an early larval stage at concentrations of paraquat that permit WT worms to develop to fertile adults (e.g., 0.2 mM). References in which this protocol was used: Van Raamsdonk and Hekimi, 2012; Van Raamsdonk et al., 2010.
       
   18. Standard output format: see Figure 1.
       
       
       Figure 1. Paraquat development assay. Results are displayed as either a graph of the furthest developmental stage reached by worms of each genotype (Left) or the average stage of development each day after transferring to paraquat plates (Right). Typically, wild-type worms can develop to fertile adults (FA) at paraquat concentrations up to 0.35 mM. Increasing concentrations of paraquat result in slower development times.
       
       
2. Acute paraquat sensitivity assay
   
   1. Prepare NGM media (see Recipes) in Erlenmeyer flask (Note 1).
      
   2. Cover the top of Erlenmeyer flask with aluminum foil and use autoclave tape to secure it in place.
      
   3. Autoclave NGM media and MgSO₄, CaCl₂ and KPI solutions (see Note 2).
      
   4. Cool media to ~55 °C (see Note 3) while stirring.
      
   5. Add MgSO₄, CaCl₂ and KPI (according to Recipes) while stirring.
      
   6. Pour control plates (no paraquat): 4 ml of media in 35 mm Petri dish (see Notes 4 and 5).
      
   7. Pour NGM media equivalent to the number of plates required plus one into a new Erlenmeyer flask or bottle. E.g., for 5 plates pour 24 ml of media into a new vessel (see Note 10).
      
   8. Add paraquat (methyl viologen) (according to Recipes) while stirring (see Notes 6 and 11).
      
   9. Pour plates: 4 ml of media in 35 mM Petri dish.
      
   10. Repeat steps B7 and B8 for each concentration of paraquat (see Note 7).
       
   11. Allow plates to solidify and dry overnight.
       
   12. 12.Seed plates with 50 µl of 10x concentrated OP50 bacteria overnight culture (see Note 12).
       
   13. Allow bacteria to and grow at room temperature for 2 days.
       
   14. Transfer 25 worms from each experimental strain to paraquat plates.
       
   15. Check the survival of the worms hourly until all worms have died (see Note 13).
       
   16. Expected results: The majority of wild-type N2 worms will die by 15 h. For young adults, 50% survival occurs around 8 h.
       
   17. Positive controls: clk-1 worms have decreased survival compared to WT, isp-1 worms have increased survival compared to WT. References in which this protocol was used: Schaar et al., 2015.
       
   18. Standard output format: see Figure 2.
       
       
       Figure 2. Acute paraquat sensitivity assay. Results are displayed as the percentage of worms that are still alive at each time point. The majority of wild-type worms do not survive past 15 h. Developing L2 and L4 worms have decreased survival compared to young adults (YA). Results represent the average of three biological replicates of 25 worms per replicate.
       
       
3. Acute juglone sensitivity assay
   
   1. Juglone plates lose their toxicity with time (Cooper et al., 2016). They need to be made fresh on the day of the assay and it is best to have a constant amount of time between pouring the plates and putting the worms on. They cannot be used for a chronic assay.
      
   2. Prepare NGM media (see Recipes) in Erlenmeyer flask (Note 1).
      
   3. Cover the top of Erlenmeyer flask with aluminum foil and use autoclave tape to secure it in place.
      
   4. Autoclave NGM media and MgSO₄, CaCl₂ and KPI solutions (see Note 2).
      
   5. Make juglone solution (see Recipes), stir in the dark for at least an hour (see Note 14).
      
   6. Cool media to ~55 °C (see Note 3) while stirring.
      
   7. Add MgSO₄, CaCl₂ and KPI (according to Recipes) while stirring.
      
   8. Pour control plates (no juglone): 10 ml of media in 60 mm Petri dishes (see Notes 4 and 5).
      
   9. Add juglone to desired concentration:
      300 μM = 2.5 ml juglone/100 ml NGM
      240 μM = 2 ml juglone/100 ml NGM
      180 μM = 1.5 ml juglone/100 ml NGM
      120 μM = 1 ml juglone/100 ml NGM
      60 μM = 0.5 ml juglone/100 ml NGM
      You can make multiple concentrations by progressively adding more juglone.
      
   10. Pour juglone plates: 10 ml of media in 60 mm Petri dishes. More concentrated plates will be a darker yellow color.
       
   11. Leave plates uncovered for 30 min to solidify agar. Plates should be in a single layer on the bench top, ideally next to a flame for sterility.
       
   12. Add 40 μl of 5x concentrated OP50 bacteria with a pipet and spread to a thin layer by tilting plate in a circle (so bacteria will dry quicker). Do not let bacteria reach the edge of the plate, to limit the number of worms that will crawl up the sides.
       
   13. Once bacteria is dry (~30 min), add 25 young adult worms per strain to the juglone plates to begin assay.
       
   14. Check worm survival periodically (e.g., 1, 2 and 4 h).
       
   15. Expected results: At 240 μM, wild-type N2 worms will start to die at 2 h and most will be dead by 4 h. Increasing juglone concentration to 300 μM results in more rapid death, while decreasing juglone concentration results in increased survival.
       
   16. Positive controls: sod-1 worms have decreased survival compared to WT. References in which this protocol was used: Van Raamsdonk and Hekimi, 2009; 2012; Van Raamsdonk et al., 2010; Cooper et al., 2015; Schaar et al., 2015; Dues et al., 2016; Machiela et al., 2016.
       
   17. Standard output format: see Figure 3.
       
       
       Figure 3. Acute juglone sensitivity assay. Results are displayed as the percentage of worms that are still alive at each time point. There is a dose-dependent decrease in survival with increasing juglone concentrations. Results represent the average of three biological replicates of 25 worms per replicate.
       
       
4. Chronic paraquat assay during adulthood
   
   1. Prepare NGM media (see Recipes) in Erlenmeyer flask (Note 1).
      
   2. Cover the top of Erlenmeyer flask with aluminum foil and use autoclave tape to secure it in place.
      
   3. Autoclave NGM media and MgSO₄, CaCl₂ and KPI solutions (see Note 2).
      
   4. Cool media to ~55 °C (see Note 3) while stirring.
      
   5. Add MgSO₄, CaCl₂ and KPI (according to Recipes) while stirring.
      
   6. Add FUdR to a concentration of 100 µM (see Note 15).
      
   7. Pour control plates (no paraquat): 10 ml of media in 60 mm Petri dishes (see Notes 4 and 5).
      
   8. Add paraquat (methyl viologen) (according to Recipes) while stirring (see Note 6).
      
   9. Pour paraquat plates: 10 ml of media in 60 mm Petri dishes.
      
   10. Repeat steps D7 and D8 for each concentration of paraquat (see Note 16).
       
   11. Allow plates to solidify and dry overnight.
       
   12. Seed plates with 150 µl of 10x concentrated OP50 bacteria overnight culture (see Note 12).
       
   13. Allow bacteria to and grow at room temperature for 2 days.
       
   14. Transfer 25 worms from each experimental strain to control and paraquat plates.
       
   15. Check the survival of the worms daily until all worms have died.
       
   16. Transfer worms to new plates every seven days.
       
   17. Expected results: Wild-type N2 worms should have an average survival of about 10 days.
       
   18. Positive controls: sod-1 and sod-2 worms have decreased survival compared to WT (Figure 4).
       References in which this protocol was used: Van Raamsdonk and Hekimi, 2009; 2012; Dues et al., 2016; Schaar et al., 2015.
       
   19. Standard output format: see Figure 4.
       
       
       Figure 4. Chronic paraquat assay. Results are typically shown as a Kaplan-Meier survival plot. Results represent the mean survival rate from a minimum of three biological replicates of 25 worms per replicate.
       

Data analysis

In the paraquat development assay, acute paraquat sensitivity assay and acute juglone sensitivity assay, we normally assess statistical significance using a two-way ANOVA with Bonferroni post-test. For the chronic paraquat assay, we assess significance using the Log-rank test. GraphPad Prism software is used to prepare all graphs and perform data analysis. Assays are performed such that the experimenter is blinded to the genotypes of the strains being tested. We perform a minimum of three independent biological replicates of at least 25 worms per strain. Worms that die due to internal hatching of progeny, externalization of internal organs, or crawling up the side of plates are censored.

Notes

1. Be sure not to overfill flask. If liquid is too near to the top it can spill over during autoclaving leading to lost volume. With small volumes, glass bottles can be used instead of Erlenmeyer flasks.
   
2. We typically use a 45 min sterilization cycle. Total time in autoclave is approximately 1 h and 15 min.
   
3. For 2 L, we normally cool for 45 min. Smaller volumes will cool more rapidly, so it is necessary to adjust cooling times.
   
4. We pour the control plates and oxidative stress plates from the same batch of media. As a result the only difference in the plates is the compound added. If we are making multiple concentrations, we make all of the concentrations from the same batch of media by adding additional amounts of the compound (e.g., paraquat or juglone) to prepare the higher concentrations.
   
5. To keep the volumes precise, we prefer to use a pipetman to ‘hand pour’ the plates. We add 10 ml to each 60 mm plate.
   
6. We make up a 1 M paraquat solution for the paraquat development assay and the chronic paraquat sensitivity assay. This solution can be stored at 4 °C for many months.
   
7. In the paraquat development assay, we normally test multiple concentrations of paraquat: 0.1 mM, 0.2 mM, 0.3 mM and 0.4 mM. Wild-type worms should be able to develop to fertile adults at a concentration of 0.35 mM but not at higher concentrations.
   
8. In the paraquat development assay, we typically transfer at least 50 eggs. For strains with markedly different embryonic development time, this assay can be adapted to look at development to adulthood beginning with L1 worms. In that case, transfer 200-300 eggs from each strain to an NGM plate. 3 h later pick L1 worms to plates containing paraquat.
   
9. For the paraquat development assay we typically use the furthest developmental stage attained (L1, L2, L3, L4, adult, fertile adult) as the outcome measure. A figure showing the different developmental stages of C. elegans can be found in the wormatlas (see Figure 6 at http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm). The furthest developmental stage attained can be measured as the average for the population of worms, the maximum or the minimum, since there can be variability within a strain, especially close to its threshold. For a more quantitative approach, determine the percentage of worms from each strain that reach adulthood. It is also possible to record the average developmental stage of the population of worms each day to compare rate of development.
   
10. Because of the high concentration of paraquat used in the acute paraquat sensitivity assay we use small 35 mm plates to minimize the amount of paraquat required. In addition, we only use a small excess of media again to minimize the amount of paraquat required. Due to the small volumes of media used, it can cool and solidify quickly. As a result it is important to pour these plates as quickly as possible. Alternatively, one could keep the media in a 55 °C water bath or on a hotplate to prevent solidification.
    
11. For the acute paraquat sensitivity assay, we make a concentrated paraquat stock solution of 3.33 M to minimize changes in volume when added to the NGM media. We add 1,167 μl of water to 1 g of paraquat powder.
    
12. To concentrate bacteria, we centrifuge at 2,935 x g (5,000 RPM) for 10 min in a 15 ml or 50 ml conical flask. Supernatant is removed and then the bacteria are resuspended by vortexing.
    
13. In the acute paraquat sensitivity assay, for some developmental stages and certain strains, we have observed worms surviving past 15 h. In this case, we stop the assay at 15 h as this is typically sufficient to observe a difference, if there is one. However, it is possible that when comparing two very resistant strains that the assay may need to be extended past 15 h.
    
14. The ease with which juglone goes into solution varies with the batch. Typically, juglone takes a long time to dissolve. We stir the juglone in ethanol for at least 1 h (while the media is autoclaving and cooling). We will normally weigh out ~0.05 g juglone and mix with 100% ethanol (23.926 ml ethanol/0.05 g juglone). Cover with foil while stirring since juglone is light sensitive.
    
15. Paraquat causes an increased rate of internal hatching of progeny. To prevent this, we add FUdR (5-fluorodeoxyuridine), which inhibits the development of progeny. It should be noted that this compound has been shown to affect the lifespan of specific genetic mutants (Van Raamsdonk and Hekimi, 2011).
    
16. In the chronic paraquat sensitivity assay, we typically test worms at 4 mM paraquat. We have also tested worms at lower concentrations such as 2 mM but this increases the duration of the assay.
    
17. Additional general notes
1. Always include all strains that you want to compare in each assay. There will be some variability in the results depending on the batch of paraquat/juglone and how fresh the plates are. It is not appropriate to compare experimental strains tested in one assay with control samples tested in a different assay.
2. All assays are performed at 20 °C. Worms are kept in 20 °C incubators during the assay and only taken out of the incubator for scoring.
3. To determine whether a worm is alive or dead, we start by simply observing the worm. If the worm is not moving, we tap the tail of the worm. If the worm doesn’t respond to the tail tap, we gently lift the head of the worm and let it fall to the agar. If the worm still doesn’t move, it is recorded as dead and removed from the plate.
4. Worms that crawl up the side of the plate, exhibit internal hatching of progeny or externalization of internal organs are censored.
   

Recipes

1. 0.5% cholesterol
   Dissolve 1 g of cholesterol in 200 ml of 100% ethanol
   Do not autoclave
   Store at room temperature
   
2. 1 M MgSO₄
   Dissolve 246.47 g MgSO₄ in 1 L of dH₂O
   Autoclave
   Store at room temperature
   
3. 1 M CaCl₂
   Dissolve 147.01 g CaCl₂ in 1 L of dH₂O
   Autoclave
   Store at room temperature
   
4. KPI
   Dissolve 35.6 g K₂HPO₄ and 108.3 g KH₂PO₄ to 1 L of dH₂O
   Adjust pH to 6.0
   Autoclave
   Store at room temperature
   
5. Nematode growth medium (NGM)
   
6. 1 M paraquat
   Add 3.89 ml of dH₂O to 1 g of paraquat
   Do not autoclave
   Store at 4 °C
7. 3.33 M paraquat
   Add 1,167 μl of dH₂O to 1 g of paraquat
   Do not autoclave
   Store at 4 °C
8. 0.1 M FUdR stock solution
   Dissolve 1 g FUdR in 40.62 ml dH₂O
   Do not autoclave
   Store in -80 °C freezer in aliquots
9. Juglone stock solution (12 mM)
   Dissolve 0.05 g of juglone in 23.93 ml of 100% ethanol
   Stir in dark for at least 1 h
   Do not autoclave
   Use immediately as toxicity is lost with time
10. 2 YT medium
    Dissolve 16 g tryptone, 10 g yeast extract and 5 g NaCl in 1 L of dH₂O
    Adjust pH to 7.0
    Autoclave
    Store at room temperature
    

Acknowledgments

This work was supported by the Van Andel Research Institute. Many other researchers have utilized similar protocols to test sensitivity to oxidative stress. These protocols are the way that we measure sensitivity to oxidative stress in our lab.

References

1. Castello, P. R., Drechsel, D. A. and Patel, M. (2007). Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain. J Biol Chem 282(19): 14186-14193.
   
2. Dues, D. J., Andrews, E. K., Schaar, C. E., Bergsma, A. L., Senchuk, M. M. and Van Raamsdonk, J. M. (2016). Aging causes decreased resistance to multiple stresses and a failure to activate specific stress response pathways. Aging (Albany NY) 8(4): 777-795.
   
3. Cooper, J. F., Dues, D. J., Spielbauer, K. K., Machiela, E., Senchuk, M. M. and Van Raamsdonk, J. M. (2015). Delaying aging is neuroprotective in Parkinson’s disease: a genetic analysis in C. elegans models. npj Parkinson's Disease 1: 15022.
   
4. Cooper, J.F., Dues, D.J., and Van Raamsdonk, J.M. (2016). Measuring sensitivity to oxidative stress: the superoxide-generator juglone rapidly loses potency with time. Worm Breeder's Gazette.
   
5. Keith, S. A., Amrit, F. R., Ratnappan, R. and Ghazi, A. (2014). The C. elegans healthspan and stress-resistance assay toolkit. Methods 68(3): 476-486.
   
6. Machiela, E., Dues, D. J., Senchuk, M. M. and Van Raamsdonk, J. M. (2016). Oxidative stress is increased in C. elegans models of Huntington's disease but does not contribute to polyglutamine toxicity phenotypes. Neurobiol Dis 96: 1-11.
   
7. Schaar, C. E., Dues, D. J., Spielbauer, K. K., Machiela, E., Cooper, J. F., Senchuk, M., Hekimi, S. and Van Raamsdonk, J. M. (2015). Mitochondrial and cytoplasmic ROS have opposing effects on lifespan. PLoS Genet 11(2): e1004972.
   
8. Van Raamsdonk, J. M. and Hekimi, S. (2009). Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans. PLoS Genet 5(2): e1000361.
   
9. Van Raamsdonk, J. M. and Hekimi, S. (2011). FUdR causes a twofold increase in the lifespan of the mitochondrial mutant gas-1. Mech Ageing Dev 132(10): 519-521.
   
10. Van Raamsdonk, J. M. and Hekimi, S. (2012). Superoxide dismutase is dispensable for normal animal lifespan. Proc Natl Acad Sci U S A 109(15): 5785-5790.
    
11. Van Raamsdonk, J. M., Meng, Y., Camp, D., Yang, W., Jia, X., Benard, C. and Hekimi, S. (2010). Decreased energy metabolism extends life span in Caenorhabditis elegans without reducing oxidative damage. Genetics 185(2): 559-571.