Oxiselect hydrogen peroxide peroxidase assay kit

Using the commercially available OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs, Inc.; San Diego, CA), hydrogen peroxide levels were measured in mouse cortical tissue collected 4 hrs following CCI-TBI or from naive and sham controls. Mice were euthanized by isoflurane overdose and decapitated to quickly remove the brain. On ice, the ipsilateral cortex was isolated and homogenized using a 2 ml Dounce manual homogenizer (Sigma-Aldrich; St. Louis, MO) in 200 μl tissue lysis buffer CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich; St. Louis, MO). Homogenate was centrifuged at 10,000 × g for 5 min at 4 °C, and supernatant was transferred to a new tube on ice. Sample aliquots were combined with the provided ADHP/HRP probe, incubated at room temperature for 30 min and read on a Spectramax M5 fluorescence plate reader (Molecular Devices; Sunnyvale, CA) (excitation 535 nm, emission 595 nm, auto cut-off 590 nm). Samples were read in duplicate and data are presented as relative fluorescent units (RFU) mean ± S.D. with n = 4 samples per condition.

The H₂O₂ assay was performed according to a previously described method [19 (link)] using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) and Horseradish peroxidase (HRP) reagents that react with H₂O₂ to emit fluorescence. Measurements were performed according to the instructions of the OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). An ADHP/HRP Working Solution was prepared by adding 100 µM ADHP and 0.2 U/mL HRP in Assay Buffer. Fifty µL UVA-exposed samples, 50 µL ADHP/HRP working solution, and 50 µL of assay buffer were transferred to 96-well plates, and the mixtures were allowed to stand at room temperature for 30 min while being protected from light. The fluorescence intensity of each mixture after incubation was measured using a microplate reader (excitation: 415 nm, emission: 610 nm).

The NO and H₂O₂ concentration in the kidney were measured using a QuantiChrom Nitric Oxide Assay Kit (BioAssay Systems, Hayward, CA, USA) and an OxiSelect Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs, San Diego, CA, USA), respectively, in accordance to the manufacturer’s protocol.

Intracellular accumulation of Reactive oxygen species (ROS) was monitored using OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs, Inc., San Diego, CA). To investigate ROS generation, the tissues and cells were homogenized in 1X Assay Buffer. The lysates were assayed according to manufacturer's procedure.

H₂O₂ concentration was measured in cell lysates using assay STA-844 (“OxiSelect Hydrogen Peroxide/Peroxidase Assay Kit”, Cell Biolabs San Diego, USA) according to manufacturer’s protocol and protein-normalized.