Sensitive H2O2 Assay via eFOX Method

The H₂O₂ assay was determined using the eFOX reagent (Cheeseman et al. 2006 (link)). The modified ferrous ammonium sulfate/xylenol orange (FOX) assay was preferred because of its sensitivity, stability, and adaptability to a large number of samples. Homogenization was performed using ice-cold acetone containing 25 mM H₂SO₄. Then samples were centrifuged for 5 min at 3000 × g at 4 °C. For 50 μl of the supernatant, 950 μl (250 μM ferrous ammonium sulfate, 100 μM sorbitol, 100 μM xylenol orange, 1% ethanol, v/v) of eFOX reagent was used. The reaction mixtures were incubated at room temperature for 30 min and absorbance at 550 and 800 nm was determined. H₂O₂ concentrations were calculated via a standard curve prepared with known concentrations of H₂O₂.