H₂O₂ Release Assay

Materials and Reagents

1. Six round bottom 13 × 100 mm glass tubes (Fisher Scientific, catalog number: 14-961-27)

2. 96-well plate, flat-bottom & black (Corning, catalog number: 3916)

3. 24-well tissue culture-treated plate (Corning, catalog number: 3524)

4. Hank’s balanced salt solution (HBSS) (ThermoFisher, catalog number: 14025092)

5. HBSS, no calcium, no magnesium, no phenol red (Gibco, catalog number: 14175095)

6. Homovanillic acid (HVA) (Sigma, catalog number: H1252)

7. Horseradish peroxidase (HRP), Type VI (Sigma, catalog number: P8375)

8. H₂O₂, 30% solution (Sigma, CAS No.: 7722-84-1)

9. IMR-90 (Coriell Institute)

10. Dulbecco's Modified Eagle Medium (DMEM) (Corning, catalog number: MT15013CV)

11. Fetal bovine serum (FBS) (Thermo Fisher, catalog number: A3160402)

12. Penicillin-Streptomycin (Thermo Fisher, catalog number: 15140122)

13. L-Glutamine (Fisher Scientific, catalog number: 25030-081)

14. Trypsin (Fisher Scientific, catalog number: MT25053CI)

15. Transforming growth factor-β1 (TGF-β1) (R&D Systems, catalog number: 101B1)

16. Hemocytometer for cell counting (Fisher Scientific, catalog number: NC1587539)

17. Homovanillic acid (HVA) stock solution, 10 mM (see Recipes)

18. Horseradish peroxidase (HRP), 5,000 U/mL in Ca²⁺ and Mg²⁺ free buffer (see Recipes)

19. H₂O_2, 1 mM (see Recipes)

20. NaOH-EDTA alkalization solution (see Recipes)

21. Assay media (see Recipes)

Equipment

1. Modular multimode microplate reader (Synergy H1, BioTek, model: H1M)

2. High-speed centrifuge (Eppendorf, model: 5430R)

3. Fisher Vortex Genie 2 (Fisher Scientific, catalog number: 02215360)

Software

1. GraphPad Prism 9.1.2 (https://www.graphpad.com)

Procedure

Cell-based assay

Day 1: Seed cells (~50,000 cells/well) in a 24-well tissue culture plate, in complete DMEM media with 10% FBS, 1% Penicillin-Streptomycin, and 2.5 mM L-Glutamine; incubate at 37°C in a humidified incubator with 5% CO₂.

Day 2: Serum-starve cells by reducing FBS concentration to 0.05% overnight (~16 h).

Day 3: In some cases, stimulation with a cytokine, such as activated TGF-β1 (2 ng/mL), may be added to induce enzyme(s) that generate extracellular H₂O₂ for variable durations of time.

Day 3 or 4: Assay for H₂O₂ release (see Figure 1).

1. Aspirate media from all wells, and wash gently with 1 mL of HBSS without phenol red.

2. Add 0.5 mL of assay media to each well, including three blank wells containing no cells.

3. Incubate at 37°C in the CO₂ incubator for 1–2 h (“t” in minutes, see step #2 under “Data analysis”).

4. At the end of the incubation period, transfer the cell-free supernatant to individual 13 × 100 mm glass tubes; add HBSS to the remaining cells for cell counting (to determine the number of cells/well, “n” in millions; see step #2 under “Data analysis”).

5. Add 1.1 µL of NaOH-EDTA alkalization solution to the glass tubes containing assay media; vortex, and allow to sit for 5 min.

6. Transfer 300 µL of alkalized assay medium from this into a 96-well black bottom plate; measure fluorescence (relative fluorescence units, RFU) at excitation and emission wavelengths of 321 nm and 421 nm, respectively.

   
   

Standard curve

1. Add 1 mL of the same assay medium (used above for cell incubation) in six 13 × 100 mm glass tubes.

2. Standard concentrations of H₂O₂ (0–5 µM) can be made by adding 1:1,000 dilutions to each of the tubes in step #1 above, including a blank (no H₂O₂).

3. The samples are vortexed, and the reaction allowed to proceed at room temperature for 5 min.

4. Add 2.2 µL of NaOH-EDTA solution to each 1 mL tube (in step #2), vortex again and sit for 5 min (together with cell-based samples in step #5 above, under “Cell-based assay”).

5. Transfer 300 µL from each standard concentration to a 96-well plate, and read fluorescence as described in step #6 above (under “Cell-based assay”).

Data analysis

1. Plot the RFU of the standards using GraphPad Prism (https://www.graphpad.com) to obtain a linear standard curve; determine the slope (“m”) (see Figure 2).

2. The rate of H₂O₂ release can then be calculated based on the following equation:

   H₂O₂ release rate (in picomoles/min/10⁶ cells):

   

   where, m = slope of standard curve

   t = duration of cell incubation (in minutes)

   n = number of cells/well (in millions)

   CF = conversion factor (0.5 × 10³ to obtain results in pmoles/min/10⁶ cells)

   [μmoles/L (μM) × 0.5 mL (reaction volume) × 10⁶pmoles/μmoles × 10^-3 L/mL]

   
   

   
   Figure 2. Standard curve for fluorometric assay for H₂O₂. Known concentrations of H₂O₂ (0–5 µM) are added to the assay media for 5 min of reaction time at room temperature; the pH of the reaction mixture is alkalinized, and fluorescence measured as described in the detailed protocol. In the example above, Y = m*X + c, where slope of the curve m = 9474, slope of the curve, and y intercept c = 66377, y intercept; in our experience, the y intercept represents “background” fluorescence that is similar to the value obtained with blanks (as described in the protocol).

   
   

Recipes

1. Homovanillic acid (HVA) stock solution, 10 mM

   Reagent Amount	Final concentration
   Homovanillic acid 91.1 mg	10 mM
   HBSS 50 mL	n/a
   Wait until fully dissolved by gentle warming if needed. Make 0.5-mL aliquots in Eppendorf tubes, and store at -80°C.




2. Horseradish peroxidase (HRP), 5000 U/mL in Ca²⁺ and Mg²⁺ free buffer

   Reagent Amount	Final concentration
   Horseradish peroxidase 25,000 U	5,000 U/mL
   HBSS 5 mL	n/a
   Store at -80°C in 50 µL aliquots.




3. H₂O₂,1 mM

   Reagent Amount	Final concentration
   H2O2, 30% solution 5.67 µL	1 mM
   H2O 50 mL	n/a
   Store at 4°C.




4. NaOH-EDTA alkalization solution

   Reagent Amount	Final concentration
   NaOH 24 g
   H2O 45 mL	n/a
   Take 45 mL of NaOH solution from above and add glycine 338 mg (0.1 M) and 329 mg of EDTA (25 mM).
   Make 1.5-mL aliquots in Eppendorf tubes and store at room temperature.




5. Assay media

   Reagent Amount	Final concentration
   HVA 0.5 mL HVA (10 mM stock)	100 µM
   HRP 50 µL HRP (5,000 U/mL stock)	5 U/mL
   HBSS with CaCl2 and MgCl2 50 mL	n/a
   Prepare fresh on the day of the assay.

Acknowledgments

This work was supported by U.S. National Institutes of Health grants, P01 HL114470, R01 HL139617, R01 HL151702, and R01 HL152246; and by the U.S. Department of Veterans Affairs Merit Award, I01BX003056. The protocol described here was adapted from Ruch and colleagues (Ruch et al., 1983), and by Chanda colleagues (Chanda et al., 2021).

Competing interests

The authors declare no competing interests in relation to the work described in this report.

References

1. Bedard, K. and Krause, K. H. (2007). The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87(1): 245-313.
2. Bienert, G. P. and Chaumont, F. (2014). Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim Biophys Acta 1840(5): 1596-1604.
3. Chanda, D., Rehan, M., Smith, S. R., Dsouza, K. G., Wang, Y., Bernard, K., Kurundkar, D., Memula, V., Kojima, K., Mobley, J. A., et al. (2021). Mesenchymal stromal cell aging impairs the self-organizing capacity of lung alveolar epithelial stem cells. Elife 10: e68049.
4. Hecker, L., Logsdon, N. J., Kurundkar, D., Kurundkar, A., Bernard, K., Hock, T., Meldrum, E., Sanders, Y. Y. and Thannickal, V. J. (2014). Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance. Sci Transl Med 6(231): 231ra247.
5. Hecker, L., Vittal, R., Jones, T., Jagirdar, R., Luckhardt, T. R., Horowitz, J. C., Pennathur, S., Martinez, F. J. and Thannickal, V. J. (2009). NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 15(9): 1077-1081.
6. Lambeth, J. D. (2004). NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4(3): 181-189.
7. Martyn, K. D., Frederick, L. M., von Loehneysen, K., Dinauer, M. C. and Knaus, U. G. (2006). Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal 18(1): 69-82.
8. Ruch, W., Cooper, P. H. and Baggiolini, M. (1983). Assay of H₂O₂ production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J Immunol Methods 63(3): 347-357.
9. Sies, H. and Jones, D. P. (2020). Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21(7): 363-383.
10. Thannickal, V. J. and Fanburg, B. L. (1995). Activation of an H₂O₂-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1. J Biol Chem 270(51): 30334-30338.
11. Thannickal, V. J. and Fanburg, B. L. (2000). Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279(6): L1005-1028.
12. Veal, E. A., Day, A. M. and Morgan, B. A. (2007). Hydrogen peroxide sensing and signaling. Mol Cell 26(1): 1-14.
13. Waghray, M., Cui, Z., Horowitz, J. C., Subramanian, I. M., Martinez, F. J., Toews, G. B. and Thannickal, V. J. (2005). Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts. FASEB J 19(7): 854-856.
14. Winterbourn, C. C. (2013). The biological chemistry of hydrogen peroxide. Methods Enzymol 528: 3-25.