Determination of H₂O₂ Generation by pHPA Assay   

Materials and Reagents

1. Nunc^TM 96-well black bottom plate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 137101 )
   
2. Eppendorf tubes
   
3. 15 and 50 ml conical tubes
   
4. Glucose (Sigma-Aldrich, catalog number: G7528 )
   
5. HEPES (1 M) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630-080 )
   
6. Sodium bicarbonate (NaHCO₃) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S233-500 )
   
7. 4-hydroxyphenylacetic acid (pHPA) (Sigma-Aldrich, catalog number: H50004 )
   
8. HRP (Sigma-Aldrich, catalog number: P8125 )
   
9. Ca²⁺/Mg²⁺/phenol red-free HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175-095 )
   
10. α-Ketoglutaric acid (Sigma-Aldrich, catalog number: K2000 )
    
11. Hydrogen peroxide solution (H₂O₂) (30%, w/w) (Sigma-Aldrich, catalog number: H1009 )
    
12. Tris
    
13. EDTA
    
14. Sucrose
    
15. Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
    
16. Phosphatase inhibitors (EMD Millipore, catalog number: 524625 )
    
17. Antimycin A (optional)
    
18. MitoTempo (optional)
    
19. pHPA buffer (see Recipes)
    
20. 1 mM H₂O₂ working stock solution (see Recipes)
    
21. Mitochondrial buffer (see Recipes)
    

Equipment

1. Kontes pellet pestle motor
   
2. Centrifuge
   
3. Plate reader (Molecular Devices, model: M2 SpectraMax )
   

Procedure

1. Prepare pHPA buffer (see Recipes)
   
2. Prepare H₂O₂ standard curve (see Figure 1)
   Note: Prepared in pHPA buffer prior to the assay (The buffer cannot be stored for subsequent measurements, make fresh at time of use).
   
1. 8-12 standards in duplicate or triplicate.
   
2. Load standards into 96-well plate.
   Highest concentration of H₂O₂: 2 µM (2 µl of 1 mM H₂O₂ working stock solution/ml pHPA buffer)
   Serial dilution: 50%
   #wells/standard: 2 or 3
   µl/well: 200
    
   
   Figure 1. Schematic of 96-well plate with 12 standards (in duplicate) and samples (in triplicate)
   
   
3. Prepare samples
   
1. Sample isolation
   
1. Samples can be whole cells, freshly isolated mitochondria or membrane fractions from various cell types (i.e., macrophages, fibroblasts, bronchoalveolar lavage cells, etc.).
   
2. Mitochondrial isolation:
   1) Lyse 10 million macrophages in (100 µl) mitochondrial buffer (see Recipes). Lysis is performed using Kontes Pellet Pestle Motor for 30 sec to homogenize each sample.
   2) Centrifuge at 2,000 x g for 10 min at 4 °C, save supernatant at 4 °C.
   3) Pellet is lysed and centrifuged [repeat steps 1) and 2)].
   4) The two supernatants are pooled and centrifuged at 12,000 x g for 15 min at 4 °C.
   5) The pellet is resuspended in (50 µl) mitochondrial buffer without sucrose [same as in 1) but do not add sucrose].
   
2. Each sample should be run in duplicate or triplicate.
   
3. Load samples into 96-well plate:
   
1. Use 5-20 µg protein or 50,000-100,000 cells per well
   
2. Suspend sample in pHPA buffer, mix well
   
3. Load 200 µl/well in duplicate
   
4. Positive controls may be added (i.e., treat cells with 100 μM antimycin A).
   
5. Negative controls may be added (i.e., treat cells with 10 μM MitoTempo).
   
4. Run assay in plate reader
   
1. Pre-warm plate reader to 37 °C.
   
2. Insert plate.
   
3. Run kinetic assay, typically for 4 h with a read every 5-10 min.
   
4. Use an excitation wavelength of 320 nm and emission of 400 nm.

Data analysis

1. Use initial readings of standards to generate a standard curve.
   
2. Determine the H₂O₂ concentration in samples by applying a linear regression to the standard curve and extrapolate.
   
3. Normalize samples to protein or cell number. Samples can be lysed in protein sample buffer to determine cellular protein levels, or alternatively when using live cells, the number of cells can be used to normalize data.
   
4. Data can be expressed as a rate over a set time (Figure 2A) or by showing a kinetic curve (Figure 2B); also see Larson-Casey et al., 2016.
   
   
   Figure 2. Mitochondrial H₂O₂ generation in macrophages expressing empty or constitutively active Akt1 (Akt1_CA) expression vectors
   

Notes

1. This assay is typically performed on freshly isolated mitochondrial (see Recipes for protocol) or membrane factions.
   
2. Prepare all working solutions on day of use and do not reuse.

Recipes

1. pHPA buffer
   50 ml total volume made up in Ca²⁺/Mg²⁺/phenol red-free HBSS
   Make up buffer in 50 ml conical tube and protect from light
   
   
2. 1 mM H₂O₂ working stock solution
   1.13 µl 30% (w/w) H₂O₂ stock solution/10 ml H₂O
   Note: H₂O₂ MW = 84.01, 30% (w/w) (H₂O₂ stock solution) equivalent to 8.82 M.
   
3. Mitochondrial buffer
   10 mM Tris, pH 7.8
   0.2 mM EDTA
   320 mM sucrose
   1 protease inhibitor tablet
   Phosphatase inhibitors diluted 1:100
   

Acknowledgments

This work was supported by 2R01ES015981 & VA merit review BX001135. This protocol was originally adapted from Panus et al. (1993).

References

1. He, C., Murthy, S., McCormick, M. L., Spitz, D. R., Ryan, A. J. and Carter, A. B. (2011). Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H₂O₂ generation. J Biol Chem 286(17): 15597-15607.
   
2. Larson-Casey, J. L., Deshane, J. S., Ryan, A. J., Thannickal, V. J. and Carter, A. B. (2016). Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity 44(3): 582-596.
   
3. Larson-Casey, J. L., Murthy, S., Ryan, A. J. and Carter, A. B. (2014). Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis. J Biol Chem 289(52): 36204-36219.
   
4. Murthy, S., Ryan, A., He, C., Mallampalli, R. K. and Carter, A. B. (2010). Rac1-mediated mitochondrial H₂O₂ generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1. J Biol Chem 285(32): 25062-25073.
   
5. Panus, P. C., Radi, R., Chumley, P. H., Lillard, R. H. and Freeman, B. A. (1993). Detection of H₂O₂ release from vascular endothelial cells. Free Radic Biol Med 14(2): 217-223.