Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans

Figure 1. Conversion of Amplex Red to Resorufin in the presence of H₂O₂ and horseradish peroxidase (HRP)

Materials and Reagents

1. 96-well microplate, black, clear bottom with lid (Corning costar, catalog number: 3603)
2. 60 mm Petri dish (Fisher Scientific, catalog number: FB0875713A)
3. 1.7 ml microcentrifuge tube
4. 15 ml conical tube
5. Micropipette tips
6. PluriStrainer 10 μm (pluriSelect, catalog number: 43-50010-01)
7. Serological pipettes
8. Caenorhabditis elegans wild-type N2 strain (Caenorhabditis Genetics Center, University of Minnesota)
9. Escherichia coli OP50 strain (Caenorhabditis Genetics Center, University of Minnesota)
10. Enterococcus faecalis OG1RF strain (ATCC 47077) 
11. Autoclaved distilled water
12. Cholesterol (Sigma-Aldrich, catalog number: C-8503)
13. Absolute ethanol (Fisher, catalog number: BP2818-4)
14. Sodium chloride (NaCl) (Fisher, catalog number: S271-500)
15. Peptone (Becton, Dickinson and Company, catalog number: 211677)
16. Agar (Becton, Dickinson and Company, catalog number: 214530)
17. Calcium chloride (CaCl₂) (EMD, catalog number: 3000)
18. Magnesium sulfate (MgSO₄) (EMD, catalog number: MX0070-1)
19. Potassium phosphate, dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: P3786)
20. Potassium phosphate, monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: 795488)
21. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S3264)
22. Nystatin (EMD Millipore, catalog number: 475914-1GM)
23. Gentamicin sulfate (Sigma-Aldrich, catalog number: G3632-5G)
24. Horse radish peroxidase (part of kit: Invitrogen, catalog number: A22188)
25. Dimethyl sulfoxide 
26. Amplex^® Red hydrogen peroxide/peroxidase assay kit (Invitrogen, catalog number: A22188)
27. Hydrogen peroxide (VWR, catalog number: VW3540-2)
28. Brain heart infusion powder (Becton, Dickinson and Company, catalog number: 211059)
29. Luria-Bertani powder (Becton, Dickinson and Company, catalog number: 244620)
30. Brain heart infusion (BHI) agar plates with gentamicin (50 μg/ml), made from BHI powder with agar added. Autoclaved, cooled, gentamycin added before pouring
31. Brain heart infusion (BHI) broth, made from BHI powder and autoclaved.
32. Nematode growth medium agar plates (see Recipes)
33. M9W buffer (see Recipes)
34. 50 mg/ml gentamicin sulfate (see Recipes)
35. 10 mg/ml nystatin (see Recipes)
36. 5 mg/ml cholesterol (see Recipes)
37. Potassium Phosphate Buffer (1 M, pH 6.0) (see Recipes)
    

Equipment

1. Micropipettes
2. Fluorescence and absorbance microplate reader (Biotek, Cytation 5 multimode reader)
3. Incubators for stable temperatures; 20 °C, 25 °C, 37 °C
4. Tabletop centrifuge
5. Stereo microscope (Zeiss, Stemi 2000)
6. Vortex mixer
7. Autoclave
   

Software

1. Gen5 (Biotek, https://www.biotek.com/products/software-robotics-software/gen5-microplate-reader-and-imager-software/)
2. Microsoft Excel (Microsoft, https://products.office.com/en-us/excel)
3. GraphPad Prism (GraphPad, https://www.graphpad.com/scientific-software/prism/)
   

Procedure

1. Worm synchronization and growth
   1. Transfer 10-15 N2 wild-type adult worms from a crowded non-starved NGM plate to a fresh 100 mm NGM plate.
   2. Let the worms lay eggs overnight at 20 °C.
   3. Next day, collect all the worms in the plate in 5 ml of M9W buffer.
   4. Filter the worm suspension through a 10 μm pluriStrainer into a 50 ml conical tube. Only L1-stage worms can pass through the filter.
   5. Count the number of L1-stage worms that pass through the filter. 
   6. Put approximately 500 of L1-staged worms on a fresh 100 mm NGM agar plate with an OP50 bacterial lawn. 
   7. Allow the worms to develop to the late L3 to early L4 stage at 20 °C, about 36 h, before starting the next step at which you will need worms at the young-adult stage.
      
      
      
2. Preparation of E. faecalis plates and control E. coli plates for worm exposure 
   1. The night before the worms reach the proper stage (young-adult), start an overnight culture of E. faecalis OG1RF in 2 ml of BHI broth and E. coli OP50 in 2 ml of LB broth at 37 °C with shaking.
   2. Add 400 μl of the E. faecalis culture to 100 mm BHI agar plates containing 50 μg/ml of gentamycin and spread to create a lawn. Gentamycin prevents the growth of any E. coli transferred over with the worms, as E. faecalis is inherently resistant. Grow at 37 °C for 4-6 h. Additionally, add 400 μl of the E. coli culture to 100 mm NGM agar plates and grow at 37 °C overnight. Make one plate each for every condition you wish to test.
   3. Remove the E. faecalis plates from the incubator and allow them to cool to room temperature.
   4. Wash the worms (young-adult stage) off from the NGM agar plate by adding 5 ml of M9W buffer. Transfer the suspended worms into 15 ml conical tubes. Wash the remaining worms off the plates using extra M9W buffer, if necessary. 
   5. Either let the worms settle to the bottom of the tube or spin them down for one minute at 200 x g. Aspirate the liquid, being careful not to disturb the collected worms. Wash the worms twice with M9W buffer. Transfer worms to E. faecalis (OG1RF) or control E. coli (OP50) plates. 
   6. Let the plates incubate overnight at 25 °C (about 16 h). This is the standard temperature used when exposing C. elegans to pathogenic bacteria.
      
      
      
3. Preparation of reagents for Amplex Red Assay (enough for 20 reactions)
   1. Dilute the 5x reaction buffer that comes with the Amplex^® Red assay kit to make a total of 25 ml of working buffer. 
   2. Thaw a tube of the horse radish peroxidase (HRP) and aliquot 100 μl into 1.7 ml microcentrifuge tubes. One tube should then be aliquoted further into 20 μl volumes, also in microcentrifuge tubes. Keep one 20 μl volume out for immediate use and freeze the rest at -20 °C. Avoid repeated freezing and thawing.
   3. Using the dimethyl sulfoxide (DMSO) that comes with the kit, add 60 μl to the Amplex^® Red reagent provided in the kit (154 g) and dissolve. Aliquot the dissolved reagent into 11 μl volumes in 1.7 ml microcentrifuge tubes. Leave one tube out for immediate use and freeze the rest at -20 °C.
   4. Right before you are ready to begin the assay, add the reagents together with the following volumes: 970 μl 1x reaction buffer, 20 μl HRP, 10 μl Amplex^® Red. Mix well and keep in the dark until use.
      
      
      
4.  Amplex^® Red assay for C. elegans
   1. Wash the worms off the E. faecalis and control plates and use 1x reaction buffer to transfer.
   2. Collect the worms in 15 ml tubes suspended in reaction buffer.
   3. Let the worms settle to the bottom of the tube or spin them down for one minute at 200 x g. Aspirate the liquid, being careful not to disturb the collected worms. 
   4. Wash the worms 3-4 times in 1 ml of reaction buffer to get rid of the bacteria.
   5. For each sample, transfer the 1 ml of worms and reaction buffer to a 60 mm empty Petri plate. Cut the end of a pipette tip to make the opening larger and, looking under the stereomicroscope, suck up about 15 worms in 25 μl of reaction buffer and transfer to a well of a 96-well plate. Do this twice to obtain 30 +/- 2 worms per 50 μl of reaction buffer/well. Count animals and adjust if necessary. Repeat twice more to generate technical triplicates of the sample. 
   6. Prepare dilutions in 1x reaction buffer of H₂O₂ for the standard curve as described in the Amplex^® Red kit literature. We use 0 μM, 1 μM, 2 μM, 3 μM, 4 μM and 5 μM as final concentrations. Dispense 50 μl of 2x concentrated H₂O₂ standards to 96-well plate in duplicate. Desired final concentrations of H₂O₂ standards will be achieved after adding the same volume of Amplex^® red reagent mix. It is recommended that the H₂O₂-standard wells should be as far as possible from the sample wells in order to prevent interference of volatile H₂O₂ from high-concentration standard wells into the sample wells. 
   7. Mix the Amplex^® Red reagent, HRP and buffer (Step C4) and add 50 μl to each well of the 96-well plate with the worm samples, controls and standards. Record the time assay begins.
      
      
      
5. Reading/recording assay
   Using a microplate reader, read the fluorescence using 540 +/-20 nm excitation and 590 +/-20 nm emission, bottom optics position and “extended” gain. We use a Biotek Cytation 5 microplate reader with Gen5 software to obtain the data. Measurements are expressed as Relative Fluorescence Units (RFU). See Figure 2 for plate organization and fluorescence readings of a sample experiment designed to test the difference of H₂O₂ production by worms in the presence of non-pathogenic (E. coli OP50 strain) and pathogenic (E. faecalis OG1RF strain) bacteria as food. Take time points at 30, 60, 120 and 180 min. Generally, the 60-min reading demonstrates the best dynamic range.
   
   
   Figure 2. Sample plate organization and fluorescence readings. Row “A” has duplicates of increasing concentrations (0-5 μM) of H₂O₂ for creating the standard curve. Row “E” has technical triplicates of the samples with the worms exposed to E. coli OP50 and E. faecalis OG1RF bacteria. Note that the samples are placed in the wells relatively far from the H₂O₂ standards. The reading was taken 60 min after mixing the samples with the Amplex Red reagent.
   

Data analysis

1. Calculation of the amount of H₂O₂ produced 
   1. Calculate the refined RFU (rRFU) of the samples by subtracting the measurement of the 0 μM H₂O₂ standard (the average of the technical duplicates) from the readings. The results represent the RFU generated by the worms in the samples during the measured time period.
   2. Calculate the rRFU of the standards by subtracting the measurement of 0 μM H₂O₂ standard (background fluorescence of the reaction buffer) from the measurements of all of the standards. 
   3. Plot the rRFU data of the standards using GraphPad Prism or similar software to create a linear standard curve. See Figure 3 for a sample standard curve created using GraphPad Prism.
   4. Calculate the slope (m) and the y-intercept (b) of the standard curve equation (rRFU = m[H₂O₂] + b). GraphPad Prism software automatically calculates the values.
   5. Use the formula below to calculate the amount of H₂O₂ produced per minute for each sample. H₂O₂ production:
      
      
      
      Note: Below is the detailed derivation of the formula above from H₂O₂ concentration.
      H₂O₂ concentration in each well:
      
      
      
      Total H₂O₂ amount produced in each well with 100 μl volume:
      
      
      
      H₂O₂ production rate
      
      
      
      H₂O₂ production rate
      
      
      
      
      Figure 3. Sample standard curve and equation. A. Refined relative fluorescence units (rRFU) were plotted against concentration. B and C. Best-fit linear curve (B) and equation (C) were generated by Graphpad Prism.
      
      
      
2. Statistical analysis of the data
   

1. Each sample has three technical replicates. GraphPad Prism or similar software is used to analyze the data. Create a bar graph by averaging the H₂O₂ production (pmoles/minutes) for each sample. Error bars represent the standard error of mean (SEM) for the three replicates. See Figure 4 for a sample bar graph of H₂O₂ production in the presence of non-pathogenic and pathogenic bacteria; E. coli (OP50 strain) and E. faecalis (OG1RF), respectively.
2. The unpaired two-tailed t-test is used to test the statistical significance of the data. 
3. To verify results with independent biological replicates, the experiment should be replicated a total of at least three times on different days with independent batches of worms. In our experience, the exact amount of ROS produced by a given batch of worms on a given day differs depending on slight differences in developmental timing and other variables. Therefore, it is not feasible to average experimental values obtained on different days. However, the PATTERNS are consistent. For example, in this experiment the animals exposed to E. faecalis ALWAYS produce significantly more ROS than animals exposed to non-pathogenic E. coli. For publication purposes, a representative experiment can be shown as primary data with biological replicates added to a supplement, if necessary.
   
   
   Figure 4. Sample bar graph showing the rate of H₂O₂ production. By each sample as picomoles per minute. Error bars represent the standard error of mean (SEM) from technical triplicates of each sample (P = 0.0002).
   

Recipes

1. 50 mg/ml gentamicin sulfate
   50 mg gentamicin sulfate is dissolved in 1 ml of distilled water and stored at -20 °C
2. 10 mg/ml nystatin
   10 mg nystatin is suspended in 1 ml 100% ethanol and stored at 4 °C
3. 5 mg/ml cholesterol
   5 mg cholesterol is dissolved in 1 ml of 100% ethanol and stored at room temperature
4. Nematode growth medium (NGM) agar plates (per liter)
   
   1. Autoclave and cool to 50 °C
      3 g NaCl
      2.5 g peptone
      18 g agar
      975 ml distilled water
   2. Add following while stirring with the same order:
      25 ml of potassium phosphate buffer (1 M, pH 6.0)
      1 ml of MgSO₄ (1 M)
      1 ml of CaCl₂ (1 M)
      1 ml of nystatin (10 mg/ml)
      1 ml of cholesterol (5 mg/ml)
   3. Pour 10 ml or 25 ml of NGM into 60 mm or 100 mm Petri dish, respectively. Let plates solidify overnight
5. M9W buffer
   3 g of KH₂PO₄
   6 g of Na₂HPO₄
   5 g of NaCl
   1 L distilled water
   Autoclave
   Add 1 ml of sterile MgSO₄ (1 M)
6. Potassium Phosphate Buffer (1 M, pH 6.0) per liter
   30.2 g of K₂HPO₄
   118.2 g of KH₂PO₄
   Complete volume to 1 L with distilled water
   Autoclave and dispense in 50 ml conical tubes for future use
   

Acknowledgments

This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI076406 to D. A. G. The content is solely the responsibility of the authors and does represent the official views of the National Institutes of Health. Also acknowledged, are the previous publications from on which this protocol was based (Chavez et al., 2007 and 2009; Liu et al., 2019; Tiller and Garsin, 2014; van der Hoeven et al., 2015).

Competing interests

The authors declare no competing interests.

References

1. Aguirre, J. and Lambeth, J. D. (2010). Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals. Free Radic Biol Med 49(9): 1342-1353. 
2. Back, P., De Vos, W. H., Depuydt, G. G., Matthijssens, F., Vanfleteren, J. R. and Braeckman, B. P. (2012). Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans. Free Radic Biol Med 52(5): 850-859. 
3. Belousov, V. V., Fradkov, A. F., Lukyanov, K. A., Staroverov, D. B., Shakhbazov, K. S., Terskikh, A. V. and Lukyanov, S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4): 281-286. 
4. Chavez, V., Mohri-Shiomi, A. and Garsin, D. A. (2009). Ce-Duox1/BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infect Immun 77(11): 4983-4989. 
5. Chavez, V., Mohri-Shiomi, A., Maadani, A., Vega, L. A. and Garsin, D. A. (2007). Oxidative stress enzymes are required for DAF-16-mediated immunity due to generation of reactive oxygen species by Caenorhabditis elegans. Genetics 176(3): 1567-1577. 
6. Dikalov, S. I. and Harrison, D. G. (2014). Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20(2): 372-382. 
7. Edens, W. A., Sharling, L., Cheng, G., Shapira, R., Kinkade, J. M., Lee, T., Edens, H. A., Tang, X., Sullards, C., Flaherty, D. B., Benian, G. M. and Lambeth, J. D. (2001). Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol 154(4): 879-891. 
8. Ewald, C. Y., Hourihan, J. M., Bland, M. S., Obieglo, C., Katic, I., Moronetti Mazzeo, L. E., Alcedo, J., Blackwell, T. K. and Hynes, N. E. (2017). NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans. Elife 6: e19493. 
9. Knoefler, D., Thamsen, M., Koniczek, M., Niemuth, N. J., Diederich, A. K. and Jakob, U. (2012). Quantitative in vivo redox sensors uncover oxidative stress as an early event in life. Mol Cell 47(5): 767-776. 
10. Labuschagne, C. F. and Brenkman, A. B. (2013). Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging. Ageing Res Rev 12(4): 918-930. 
11. Liu, Y., Kaval, K. G., van Hoof, A. and Garsin, D. A. (2019). Heme peroxidase HPX-2 protects Caenorhabditis elegans from pathogens. PLoS Genet 15(1): e1007944. 
12. McCallum, K. C. and Garsin, D. A. (2016). The role of reactive oxygen species in modulating the Caenorhabditis elegans immune response. PLoS Pathog 12(11): e1005923. 
13. Miranda-Vizuete, A. and Veal, E. A. (2017). Caenorhabditis elegans as a model for understanding ROS function in physiology and disease. Redox Biol 11: 708-714. 
14. Mohanty, J. G., Jaffe, J. S., Schulman, E. S. and Raible, D. G. (1997). A highly sensitive fluorescent micro-assay of H₂O₂ release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods 202(2): 133-141. 
15. Moribe, H., Konakawa, R., Koga, D., Ushiki, T., Nakamura, K. and Mekada, E. (2012). Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans. PLoS Genet 8(9): e1002957. 
16. Pellegrino, M. W., Nargund, A. M., Kirienko, N. V., Gillis, R., Fiorese, C. J. and Haynes, C. M. (2014). Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection. Nature 516(7531): 414-417. 
17. Schulz, T. J., Zarse, K., Voigt, A., Urban, N., Birringer, M. and Ristow, M. (2007). Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab 6(4): 280-293. 
18. Sem, X. and Rhen, M. (2012). Pathogenicity of Salmonella enterica in Caenorhabditis elegans relies on disseminated oxidative stress in the infected host. PLoS One 7(9): e45417. 
19. Tiller, G. R. and Garsin, D. A. (2014). The SKPO-1 peroxidase functions in the hypodermis to protect Caenorhabditis elegans from bacterial infection. Genetics 197(2): 515-526. 
20. van der Hoeven, R., Cruz, M. R., Chavez, V. and Garsin, D. A. (2015). Localization of the dual oxidase BLI-3 and characterization of its NADPH oxidase domain during infection of Caenorhabditis elegans. PLoS One 10(4): e0124091.