Amplex red catalase assay kit

UV-Vis transmittance spectra were measured from 200 to 1100 nm. Heating curves were recorded by an IR thermal camera (FLIR system, ThermaCAM Researcher). The film coloration and bleaching data were recorded under irradiation by a simulated solar AM1.5G lamp with an average irradiance of 153 mW/cm². The cold environment is created by ice pack. The electron spin resonance (ESR) spectra were recorded at 77 K using a JEOL JES-FA200 spectrometer. Scanning electron microscopy (SEM) images were obtained with a JEOL 6390 scanning electron microscope. The SEM samples were prepared by sintering sample powder onto a Si substrate. The amount of H₂O₂ was determined from the fluorescence intensity of oxidized Amplex^® Red (10-acetyl-3,7-dihydroxyphenoxazine). The Amplex^® Red reagent reacts with H₂O₂ in the presence of horseradish peroxidase (HRP) in a 1:1 stoichiometric ratio to form fluorescent resorufin. Here, this method was used to quantify the amount of hydrogen peroxide desorbed from various samples. The resorufin absorption was measured in a 96-well plate at 571 nm with a UV/Vis spectrophotometer (Perkin Elmer, Lambda 35). The Amplex^® Red Catalase Assay Kit was purchased from Molecular Probes (USA). For the calibration curve, the absorption data collected at 571 nm over a wide H₂O₂ concentration images were created by Mathematic software.

Two- and three-week-old plant tissues were harvested and frozen after treatment with MV (10 μM) and treatment with drought (air-drying) in the same manner as water-loss assay for 4 h, respectively. Frozen tissues were ground in liquid nitrogen. Ground tissues (50 mg) were soaked in 20 mM potassium phosphate buffer (pH 6.5) on ice. The extracts were clarified by centrifugation at 13,500g for 15 min at 4 °C. H₂O₂ concentration and peroxidase activity were measured in the supernatants using an Amplex Red hydrogen peroxide/peroxidase assay kit according to the manufacturer's instructions (Molecular Probes). Fluorescence was detected by a spectrofluorometer (Molecular Device). Catalase activity was measured with same plant extracts using the Amplex Red catalase assay kit according to the manufacturer's instructions (Molecular Probes).

Total anti-oxidant capacity (T-AOC) and enzyme activity of total superoxide dismutase (SOD) were examined by kits (Nanjing Jiancheng Bioengineering Institute, China) and activity of catalase (CAT) was determined by Amplex^® Red catalase assay kit (Molecular Probes Inc., Invitrogen, Eugene, OR, United States). Commercial Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1, resembles to human IL-8; R&D Inc., Minneapolis, MN, United States), TNF-α (eBioscience, San Diego, CA, United States), IL-10 (BD Biosciences, San Diego, CA, United States), and adiponectin (Invitrogen, Eugene, OR, United States) in rat heart homogenates according to the manufacturer’s instructions. The absorbance was measured by a microplate-reader and the results were corrected by heart protein concentration.

Catalase activity was tested using an Amplex^® Red Catalase Assay Kit (Molecular Probes, Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. The kit provides an ultrasensitive but, simple method for measuring catalase activity based on a competition assay between catalase enzyme and the Amplex Red reagent. Catalase first reacts with H₂O₂ producing H₂O and O₂. The Amplex Red then reacts with any remaining H₂O₂ in the presence of horseradish peroxidase (HRP), producing a fluorescent oxidation product, resorufin. Increased production of catalase enzyme(s) overtime reduces the resorufin signal. Overnight bacterial cultures (O.D 600 = 0.2) were centrifuged and the pellets were lysed in 0.1 M Tris buffer (pH 7.4).
The experiment was performed in a 96-well plate by adding 25 μl each of SRB02 and AY1 cell lysates, catalase standard curve samples, and 40 μM H₂O₂. Following the decomposition of hydrogen peroxide by catalase, 50 μM Amplex Red reagent and 0.2 U/ml horseradish peroxidase (HRP) were added. Fluorescence was measured using a Multiskan^™ GO microplate spectrophotometer (Thermo Fisher, Waltham, MA, USA) at 590 nm. The experiment was repeated three times and data were analyzed for significant differences through Student’s T-test at 95% level of significance using Microsoft Excel.

For analysis of SOD, CAT, and POD activities, total protein was extracted using 20 mM potassium phosphate buffer (pH 6.5) and quantitated according to the Bradford method [17 (link)]. SOD, CAT, and POD activities were analyzed using a SOD Assay Kit-WST (Sigma-Aldrich, St. Louis, MO, USA), Amplex Red Catalase Assay Kit (Molecular Probes, Eugene, OR, USA), and Amplex^™ Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, Carlsbad, CA, USA), respectively.

Cells were sonicated in 0.1 M Tris-HCl (pH7.5) for two 30-s bursts. After centrifugation, the supernatant was used to measure catalase activity with an Amplex Red catalase assay kit, according to the manufacturer’s protocol (Molecular Probes). After incubating the samples with 40 mM H₂O₂ for 30 min, the remaining H₂O₂ was measured to determine the catalase activity. Amplex Red and horseradish peroxidase react with H₂O₂ to produce resorufin, a fluorescent compound detectable by spectrophotometry. Standard curves for the enzymatic activity of catalase were determined using purified catalase. The protein concentration was measured using a BCA Protein Quantitation Analysis Kit (Solarbio). The enzyme activities were normalized by protein content of samples and expressed relative to the value in the control group.