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Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays
秀丽隐杆线虫中的氧化胁迫测定:百草枯和胡桃酮敏感性分析法   

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Abstract

Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a balance between basal levels of ROS, the ability to detoxify ROS, and the ability to repair ROS-mediated damage.

Keywords: Oxidative stress(氧化胁迫), C. elegans(秀丽隐杆线虫), Paraquat(百草枯), Juglone(胡桃酮), Stress resistance(抗逆性), Reactive oxygen species(活性氧)

Background

A number of approaches have been used to test sensitivity to oxidative stress in Caenorhabditis elegans including exposure to paraquat, juglone, t-BOOH, arsenite, H2O2, or hyperbaric oxygen(Keith et al., 2014). All of these assays serve to increase the levels of ROS in the worm to a point where survival is decreased. The assays differ in the primary type of ROS that the worm is exposed to (e.g., superoxide, hydrogen peroxide), the rate of exposure (acute versus chronic) and the subcellular compartment believed to be most affected (e.g., paraquat increases superoxide levels primarily in the mitochondria (Castello et al., 2007). Based on these differences, it is possible that a particular strain of worm exhibits increased sensitivity or increased resistance to oxidative stress in one assay, but does not show a difference in another assay. It is also possible that a strain of worms is sensitive to oxidative stress at one age, but resistant to that same form of oxidative stress at a different age. Thus, to obtain a full understanding of sensitivity to oxidative stress in a particular strain it is necessary to use multiple assays at different time points.

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 scientificTM, 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.

简介

已经提出氧化应激是老化的主要原因之一,并且已经涉及许多疾病的发病机理。对氧化应激的敏感性可以通过量化暴露于活性氧(ROS) - 生成化合物如百草枯或胡桃酮后的存活来测量。对氧化应激的敏感性是ROS的基础水平,ROS解毒能力和修复ROS介导的损伤的能力之间的平衡。

背景 已经使用许多方法来测试秀丽隐杆线虫中的氧化应激的敏感性,包括接触百草枯,胡桃碱,t-BOOH,亚砷酸盐,H 2 O 2 O 2或高压氧(Keith等人,2014)。所有这些测定用于将蠕虫中的ROS水平提高到生存降低的程度。蠕虫暴露于(例如,超氧化物,过氧化氢),暴露速率(急性与慢性)和认为受影响最严重的亚细胞区域(ROS)的主要类型不同,百草枯主要在线粒体中增加超氧化物水平(Castello et al。,2007)。基于这些差异,特定的蠕虫菌株可能表现出增加的敏感性或在一个测定中增加的抗氧化应激,但在另一个测定中没有显示出差异。蠕虫菌株也可能在一个年龄对氧化应激敏感,但在不同的时间对该氧化应激的相同形式有抗性因此,为了获得对特定菌株对氧化应激敏感性的充分了解,有必要在不同时间点使用多种测定法。

关键字:氧化胁迫, 秀丽隐杆线虫, 百草枯, 胡桃酮, 抗逆性, 活性氧

材料和试剂

  1. 培养皿60 x 15 mm 500/cs(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875713A)
  2. 培养皿35 x 10 mm 500/cs(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875711YZ)
  3. 高压胶带
  4. 铝箔
  5. 99.95%铂,0.05%铱丝(3 ft/pk)(Tritech Research,目录号:PT-9901)
  6. OP50 E。大肠杆菌细菌(明尼苏达大学,线虫遗传学中心,N/A)
  7. 实验和控制。线虫菌株(明尼苏达大学,线虫遗传学中心,N/A)
  8. 来自实验和对照的鸡蛋C。线虫株
  9. 甲基紫精二氯化物水合物(百草枯)(Sigma-Aldrich,目录号:856177)
  10. FUdR(5-氟-2'-脱氧尿苷)(Sigma-Aldrich,目录号:F0503)
  11. 5-羟基-1,4-萘醌(胡桃碱)(Sigma-Aldrich,目录号:H47003)
  12. 100%乙醇
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  14. Bacto蛋白胨(BD,目录号:211677)
  15. 琼脂(Sigma-Aldrich,目录号:A1296)
  16. 胆固醇(Sigma-Aldrich,目录号:C8667)
  17. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M1880)
  18. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C3881)
  19. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:P2222)
  20. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  21. 胰蛋白胨(Sigma-Aldrich,目录号:T7293)
  22. 酵母提取物(Sigma-Aldrich,目录号:70161)
  23. 0.5%胆固醇(见食谱)
  24. 1 M MgSO 4(参见食谱)
  25. 1 M CaCl 2 (见配方)
  26. KPI(见配方)
  27. 线虫生长培养基(NGM)(见食谱)
  28. 1百草草(见食谱)
  29. 3.33百草枯(见配方)
  30. 0.1 M FUdR储备溶液(见配方)
  31. Juglone储备溶液(12 mM)(见食谱)
  32. 2 YT培养基(见食谱)

设备

  1. 锥形瓶(Thermo Fisher Scientific,Fisher Scientific,目录号:FB5006000)
  2. M50立体显微镜(Leica,型号:10450154)
  3. Pipetor(Gilson,目录号:F167300)
  4. 高压灭菌器(Tuttnauer,型号:6690)
  5. 搅拌电热板(康宁,目录号:6795-620)
  6. 离心机(Eppendorf,型号:5430)
  7. 冷藏培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:51028064; 37-20)
  8. 本生灯(Humbolt,目录号:H5870)

软件

  1. GraphPad Prism软件(我们使用版本5.01)

程序

  1. 百草枯发育测定 - 发展期间对慢性氧化应激的敏感性
    1. 在锥形瓶中准备NGM培养基(参见食谱)(注1)
    2. 用铝箔覆盖锥形瓶的顶部,并使用高压胶带将其固定到位。
    3. 高压灭菌NGM介质和MgSO 4,CaCl 2和KPI溶液(见注2)。
    4. 搅拌下将介质冷却至〜55°C(见注3)
    5. 在搅拌下加入MgSO 4,CaCl 2和KPI(根据食谱)。
    6. 倒入对照板(无百草枯):在60毫米培养皿中10ml培养基(见注释4和5)。
    7. 在搅拌下加入百草枯(甲基紫精)(根据食谱)(见附注6)。将百草枯容许〜60秒混合到培养基中。
    8. 倒盘:在60毫米培养皿中10ml培养基。
    9. 每次浓缩百草枯重复步骤A7和A8(见注7)
    10. 允许板在室温下固化并干燥过夜。
    11. 种子板与100微升的OP50细菌过夜培养
    12. 让细菌干燥并在室温下生长2天。
    13. 转移相应数量的鸡蛋从每个被测试的菌株控制含有百草枯的板和板(注8),确保你不转移任何卵孵化到第一个幼虫发育阶段(L1蠕虫)。
    14. 检查以确保鸡蛋第二天孵出。
    15. 每天监测蠕虫的发育阶段(见注9)。
    16. 预期结果:野生型N2蠕虫应发育成高达约0.35 mM百草枯的板块上的肥沃成年人。
    17. 积极的控制: ,sod-2 都在早期幼虫阶段逮捕百草枯浓度,允许WT蠕虫发育成肥沃的成年人(例如,0.2mM)。参考文献:Van Raamsdonk和Hekimi,2012; Van Raamsdonk等人。,2010
    18. 标准输出格式:见图1.


      图1.百草枯开发试验。 结果显示为转移到百草枯平板后每个基因型蠕虫(左)或平均发育阶段达到的最远发育阶段的图表(右图)。通常,在百草枯浓度高达0.35mM的情况下,野生型蠕虫可以发育成成熟的成虫(FA)。百草枯浓度增加导致开发时间较慢。

  2. 急性百草枯敏感性分析
    1. 在锥形瓶中准备NGM培养基(参见食谱)(注1)
    2. 用铝箔覆盖锥形瓶的顶部,并使用高压胶带将其固定到位。
    3. 高压灭菌NGM介质和MgSO 4,CaCl 2和KPI溶液(见注2)。
    4. 搅拌下将介质冷却至〜55°C(见注3)
    5. 在搅拌下加入MgSO 4,CaCl 2和KPI(根据食谱)。
    6. 倒入对照板(无百草枯):在35 mm培养皿中加入4 ml培养基(见注4和5)。
    7. 将NGM介质相当于所需的板数加上一个新的锥形瓶或瓶子。对于5块板,将24毫升的介质倒入新的容器中(见注10)。
    8. 在搅拌下加入百草枯(甲基紫精)(根据食谱)(见注释6和11)。
    9. 倒入平板:在35mM培养皿中4ml培养基。
    10. 对每种浓缩百草枯重复步骤B7和B8(见注7)
    11. 让板固化并过夜干燥。
    12. 12.种子平板与50μl10x浓缩的OP50细菌过夜培养(见注12)
    13. 允许细菌在室温下生长2天。
    14. 将25个蠕虫从每个实验菌株转移到百草枯平板。
    15. 检查蠕虫的生存时间,直到所有蠕虫死亡(见附注13)。
    16. 预期结果:大多数野生型N2蠕虫将死亡15小时。对于年轻人,50%的存活发生在8小时左右。
    17. 阳性对照:与WT相比,蠕虫具有降低的存活率,与野生型相比,蠕虫具有增加的存活率。使用该协议的参考文献:Schaar等人。,2015年
    18. 标准输出格式:见图2.


      图2.急性百草枯敏感性测定。结果显示为每个时间点上仍然存在的蠕虫的百分比。大多数野生型蠕虫在15 h以后才能生存。与年轻人(YA)相比,发展L2和L4蠕虫的生存降低。结果表示每次重复的25个蠕虫的三个生物重复的平均值。

  3. 急性胡桃敏感性分析
    1. Juglone板块随着时间的推移而丧失毒性(Cooper等人,2016)。他们需要在测定的当天做新鲜,最好是在浇注板块和放入蠕虫之间有一定的时间。它们不能用于慢性测定。
    2. 在锥形瓶中准备NGM培养基(参见食谱)(注1)
    3. 用铝箔覆盖锥形瓶的顶部,并使用高压胶带将其固定到位。
    4. 高压灭菌NGM介质和MgSO 4,CaCl 2和KPI溶液(见注2)。
    5. 制作胡桃汁溶液(参见食谱),在黑暗中搅拌至少一小时(见附注14)
    6. 搅拌下将介质冷却至〜55°C(见注3)
    7. 在搅拌下加入MgSO 4,CaCl 2和KPI(根据食谱)。
    8. 倒入对照板(不含胡桃醇):在60毫米培养皿中10ml培养基(见注释4和5)。
    9. 将胡桃加入所需浓度:
      300μM= 2.5毫升胡桃碱/100毫升NGM
      240μM= 2毫升胡桃/100毫升NGM
      180μM= 1.5ml胡桃酮/100ml NGM
      120μM= 1毫升胡桃酮/100毫升NGM
      60μM= 0.5毫升胡桃碱/100毫升NGM
      您可以通过逐渐添加更多的胡桃来制造多个浓度。
    10. 倒入胡桃木板:将60毫升培养皿中的10ml培养基。更浓缩的色块将会变得更深黄色。
    11. 将板露出30分钟以固化琼脂。板应在台面上的单层中,理想地在灭菌火焰旁边。
    12. 用移液管加入40μl5x浓缩的OP50细菌,并通过在一个圆圈中倾斜板将其铺展到薄层(因此细菌会更快地干燥)。不要让细菌到达板的边缘,限制蠕虫爬行的次数。
    13. 一旦细菌干燥(约30分钟),每个菌株每年加入25只幼虫,以开始测定。
    14. 定期检查蠕虫生存(例如,,1,2和4h)。
    15. 预期结果:在240μM时,野生型N2蠕虫将在2 h时开始死亡,大部分死亡4 h。将浓度增加至300μM导致更快速的死亡,同时减少胡桃浓度导致存活率增加。
    16. 阳性对照:与WT相比,蠕虫具有降低的存活率。参考文献:Van Raamsdonk和Hekimi,2009; 2012; Van Raamsdonk等人,2010; Cooper等人,2015; Schaar等人,2015; 2016年,Dues 等。 Machiela等人。,2016。
    17. 标准输出格式:见图3.


      图3.急性胡桃灵敏度测定。结果显示为每个时间点上仍然存在的蠕虫的百分比。随着胡桃浓度的增加,存活率随剂量依赖性降低。结果表示每次重复的25个蠕虫的三个生物重复的平均值。

  4. 成年期慢性百草枯测定
    1. 在锥形瓶中准备NGM培养基(参见食谱)(注1)
    2. 用铝箔覆盖锥形瓶的顶部,并使用高压胶带将其固定到位。
    3. 高压灭菌NGM介质和MgSO 4,CaCl 2和KPI溶液(见注2)。
    4. 搅拌下将介质冷却至〜55°C(见注3)
    5. 在搅拌下加入MgSO 4,CaCl 2和KPI(根据食谱)。
    6. 将FUdR加入浓度为100μM(见附注15)
    7. 倒入对照板(不含百草枯):在60 mm培养皿中加入10ml培养基(见注释4和5)。
    8. 在搅拌下加入百草枯(甲基紫精)(根据食谱)(见附注6)。
    9. 倒入百草枯平板:将60毫升培养皿中的10ml培养基。
    10. 对每种浓缩百草枯重复步骤D7和D8(见附注16)
    11. 让板固化并过夜干燥。
    12. 用150μl10x浓缩的OP50细菌过夜培养的种子板(见附注12)
    13. 允许细菌在室温下生长2天。
    14. 将25个蠕虫从每个实验菌株转移到对照和百草枯板上
    15. 每天检查蠕虫的生存状况,直到所有的蠕虫死亡为止。
    16. 每七天将蠕虫传播给新的板材。
    17. 预期结果:野生型N2蠕虫的平均存活期应为10天左右。
    18. 与WT相比,积极的控制: sod-1 和 sod-2 蠕虫的生存降低(图4)。
      参考文献:Van Raamsdonk和Hekimi,2009; 2012; ,2016;

    19. 标准输出格式:见图4.


      图4.慢性百草枯测定。结果通常显示为Kaplan-Meier生存曲线。结果表示每次重复的25个蠕虫的最少三次生物重复的平均存活率。

数据分析

在百草枯发展测定中,急性百草枯敏感性测定和急性胡桃敏感性测定,我们通常使用Bonferroni后测试的双因素ANOVA来评估统计学显着性。对于慢性百草枯测定,我们使用对数秩检验评估显着性。 GraphPad Prism软件用于准备所有图形并执行数据分析。进行测定使得实验者对所测试的菌株的基因型不知情。我们对每个菌株进行至少25次蠕虫的至少三次独立生物复制。由于子代的内部孵化,内部器官外部化或爬行板的死亡而死亡的蠕虫被检查。

笔记

  1. 确保不要过量的烧瓶。如果液体太靠近顶部,则在高压灭菌过程中可能会溢出,导致体积减少。小批量使用玻璃瓶代替锥形瓶。
  2. 我们通常使用45分钟的灭菌周期。高压釜中的总时间约为1小时15分钟。
  3. 对于2L,我们通常冷却45分钟。更小的体积会更快地冷却,所以有必要调整冷却时间。
  4. 我们从同一批介质倒入对照板和氧化应激板。因此,添加的化合物是唯一的差异。如果我们制造多个浓度,我们通过添加额外量的化合物(例如,百草枯或胡桃)来制备来自相同批次的培养基的所有浓度,以制备更高的浓度。
  5. 为了保持体积的精确度,我们更倾向于使用移液器来"倒"。我们每个60毫米的平板加10毫升
  6. 我们为百草枯发展测定和慢性百草枯敏感性测定组成了1百万分百倍草解决方案。该解决方案可以在4°C下储存数月。
  7. 在百草枯发育测定中,我们通常测试多种浓度的百草枯:0.1mM,0.2mM,0.3mM和0.4mM。野生型蠕虫应能够发育成浓度为0.35 mM,但浓度不高的肥沃成虫
  8. 在百草枯发育测定中,我们通常会转移至少50个鸡蛋。对于具有明显不同的胚胎发育时间的菌株,该测定可以适应于从L1蠕虫开始到成年的发育。在这种情况下,将200-300个鸡蛋从每个菌株转移到NGM板上。 3小时后,将L1蠕虫挑选到含百草枯的板上。
  9. 对于百草枯发展测定,我们通常使用最远的发育阶段(L1,L2,L3,L4,成年,肥沃的成年人)作为结果测量。显示了C的不同发展阶段的数字。 elegans 可以在蠕虫病毒中找到(参见图6中的 http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm )。所发展的最远的发展阶段可以作为蠕虫群体的平均值,最大值或最小值来衡量,因为菌株中存在变异性,特别是接近其阈值。对于更定量的方法,确定到达成年的每个菌株的蠕虫百分比。也可以每天记录蠕虫群体的平均发育阶段,以比较发展的速度
  10. 由于在百草枯敏感性测定分析中使用百草枯的浓度很高,因此我们使用小型35毫米平板来最大限度地减少百草枯所需的量。此外,我们再次使用少量媒体,以尽量减少百草枯所需的量。由于使用的介质体积小,可以快速冷却和固化。因此,尽可能快地倒出这些板是非常重要的。或者,可以将介质保持在55℃的水浴或加热板上以防止固化。
  11. 对于急性百草枯敏感性测定,我们制备浓缩的百草枯储备溶液3.33 M,以便在添加到NGM培养基中时最小化体积变化。我们向1克百草枯粉末中加入1,167微升的水。
  12. 为了浓缩细菌,我们在15ml或50ml锥形瓶中以2,935×g(5,000RPM)离心10分钟。去除上清液,然后通过涡旋将细菌重新悬浮。
  13. 在急性百草枯敏感性测定中,对于一些发育阶段和某些菌株,我们观察到蠕虫在15小时内存活。在这种情况下,我们在15小时内停止测定,因为这通常足以观察差异,如果有的话。然而,当比较两种非常耐药的菌株时,可能需要在15小时以后延长测定。
  14. Juglone进入解决方案的容易程度因批次而异。通常,胡桃花很长时间才能溶解。我们在乙醇中搅拌胡桃至少1小时(当培养基进行高压灭菌和冷却时)。通常称重约0.05克胡桃碱,并与100%乙醇(23.926毫升乙醇/0.05克胡桃子)混合。搅拌时,用金箔盖上薄荷,因为胡桃是敏感的
  15. 百草枯导致子代内部孵化率提高。为了防止这种情况,我们添加了抑制后代发育的FUdR(5-氟脱氧尿苷)。应该指出,这种化合物已被证明会影响特定遗传突变体的寿命(Van Raamsdonk和Hekimi,2011)。
  16. 在慢性百草枯敏感性测定中,我们通常以4mM百草枯测试蠕虫。我们还测试了较低浓度的蠕虫,如2 mM,但这增加了测定的持续时间
  17. 附加一般注释
    1. 始终包括您想在每个测定中比较的所有菌株。结果取决于百草枯/胡桃子的批次以及板材的新鲜度会有一些变化。将在一个测定中测试的实验菌株与在不同测定中测试的对照样品进行比较是不合适的。
    2. 所有测定均在20℃进行。在测定期间将蠕虫保存在20℃的培养箱中,并且仅从培养箱中取出进行评分。
    3. 为了确定蠕虫是否活着还是死亡,我们首先要简单地观察蠕虫。如果蠕虫不动,我们点击蠕虫的尾巴。如果蠕虫对尾巴没有响应,我们轻轻地抬起蠕虫的头部,让它落到琼脂上。如果蠕虫仍然没有移动,它被记录为死亡并从板上移除。
    4. 爬行板的蠕虫,内部孵出后代或内部器官外部化被检查。

食谱

  1. 0.5%胆固醇
    将1克胆固醇溶于200毫升100%乙醇中 不要高压
    在室温下存放
  2. 1 M MgSO 4
    在1L dH 2 O溶液中溶解246.47g MgSO 4·4 / 高压灭菌器
    在室温下存放
  3. 1 M CaCl 2
    溶解在1L dH 2 O中的147.01g CaCl 2 2 高压灭菌器
    在室温下存放
  4. KPI
    将35.6g K 2 H 2 HPO 4和108.3g KH 2 PO 4溶解至1L dH 3 2 O
    调节pH至6.0
    高压灭菌器
    在室温下存放
  5. 线虫生长培养基(NGM)
  6. 百草枯1百草枯 将3.89ml dH 2 O加入到1g百草枯中 不要高压
    储存于4°C
  7. 3.33百草枯
    将1,167μldH 2 O加入到1g百草枯中 不要高压
    储存于4°C
  8. 0.1 M FUdR储备溶液
    将1g FUdR溶于40.62ml dH 2 O中 不要高压
    储存在-80°C冰箱中等分
  9. Juglone储备溶液(12 mM)
    将0.05g胡桃酮溶于23.93ml 100%乙醇中 在黑暗中搅拌至少1小时
    不要高压
    立即使用,因为毒性随时间的流失而消失
  10. 2 YT中等
    在1L dH 2 O溶液中溶解16g胰蛋白胨,10g酵母提取物和5g NaCl,
    调节pH至7.0
    高压灭菌器
    在室温下存放

致谢

这项工作得到了Van Andel研究所的支持。许多其他研究人员已经使用类似的方案来测试对氧化应激的敏感性。这些协议是我们实验室测量氧化应激敏感性的方法。

参考文献

  1. Castello,PR,Drechsel,DA和Patel,M。(2007)。  线粒体是大脑中百草枯诱导的活性氧生成的主要来源。生物化学 282(19):14186-14193。
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引用:Senchuk, M. M., Dues, D. J. and Van Raamsdonk, J. M. (2017). Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays. Bio-protocol 7(1): e2086. DOI: 10.21769/BioProtoc.2086.
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