Horseradish peroxidase

Following treatment, 100 μl samples were taken from the cell culture medium and immediately frozen at −80°C for subsequent analysis. The lactate assay was performed using 1 μl of each sample with 9 μl of water in a 96-well cell culture plate, alongside lactate standards for comparison. Approximately 190 μl of reaction mixture containing 2.5 U/L lactate oxidase (Merck-Sigma Aldrich, St Louis, MO), 50 U/L horseradish peroxidase (Merck-Sigma Aldrich, St Louis, MO), and 20 μM Amplex Red (Thermo Fisher Scientific-Invitrogen, Grand Island, NY) in 0.1 M sodium phosphate buffer (pH 7.5) was added to each well. The reactions were protected from light and incubated for 30 min at 37°C. Fluorescence was measured (Ex 520 nm, Em 580–640 nm) using a GloMax Discover system (Promega, Madison, WI). Glucose assays were performed using a similar method, however with a reaction mixture containing 135 U/L glucose oxidase (Merck-Sigma Aldrich, St Louis, MO), 50 U/L horseradish peroxidase (Merck-Sigma Aldrich, St Louis, MO), and 20 μM Amplex Red (Thermo Fisher Scientific-Invitrogen, Grand Island, NY) in 0.1 M sodium phosphate buffer (pH 6).

A 0.5 mg/mL of leucocrystal violet solution was obtained by dissolving 10 mg of leucocrystal violet (Sigma-Aldrich, St. Louis, MO, USA) in 20 mL of distilled water (DW). A 1.0 mg/mL horseradish peroxidase solution was obtained by dissolving 10 mg of horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) in 10 mL of DW. A series of H₂O₂ solutions, the concentrations of which were 0.05 μg /mL, 0.10 μg /mL, 0.25 μg/mL, 0.50 μg /mL, 1.0 μg /mL, 2.5 μg /mL, and 5.0 μg /mL, were obtained by diluting 30% H₂O₂ stock solution (Fisher Scientific, Pittsburgh, PA, USA) with DW.
To obtain a H₂O₂ solution calibration curve, 100 μL of 0.5 mg/mL leucocrystal violet solution and 50 μL of 1.0 mg/mL horseradish peroxidase solution were added into 100 μL, 50 μL, 20 μL, 10 μL, 5 μL, 2.5 μL, and 1.25 μL of H₂O₂ solutions and then diluted to 10 mL with acetate buffer (pH = 4.5). The concentrations of H₂O₂ in these solutions were 5.0 μg /mL, 2.5 μg/mL, 1.0 μg /mL, 0.5 μg /mL, 0.25 μg /mL, 0.1 μg /mL, and 0.05 μg /mL respectively. After vibrated for 30 s, these solutions were measured with an Ultraviolet-visible (UV-Vis) spectrophotometer (UV-2600, Shimadzu Co., Kyoto, Japan) at a wavelength of 596 nm against a reference prepared using the same method but with no hydrogen peroxide. A calibration curve was thus obtained [16 (link)].

Mitochondria were suspended in buffer B in a 1 ml cuvette inside a spectrophotometer (QM-8; Photon Technology International (PTI), Birmingham, NJ, USA). The rate of H₂O₂ release was measured using Amplex red (12.5 μM; Molecular Probes, Eugene, OR, USA) and horseradish peroxidase (0.1 U/ml; Sigma) at excitation and emission wavelengths of 530 and 583 nm, respectively. H₂O₂ is the direct product of O^•−₂ when catalyzed by O^•−₂ dismutase (SOD) in the absence of nitric oxide. H₂O₂ levels were calibrated over a range of 10–200 nM H₂O₂ (Sigma) added to buffer B in the absence of mitochondria and in the presence of Amplex red and horseradish peroxidase. Mitochondrial volume change (increase/decrease) was assessed by monitoring changes in 90° light scattering at an excitation and emission wavelength of 520 nm inside the same cuvette-based PTI.