LPS, Apelin-13, F13A (a selective APJ receptor antagonist), DPI (an NAPDH oxidase inhibitor, diphenylene iodonium), NAC (a superoxide inhibitor, N-Acetyl Cysteine) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Mouse tumor necrosis factor (TNF)-α, IL-1β, IL-6 and Apelin-13 enzyme linked immunosorbent assay (ELISA) kits were purchased from Cusabio (Houston, TX, USA). The reactive oxygen species assay kit (DCF-DA) was from Applygen (Beijing, China). The H2O2 assay kit was from Beyotime (Shanghai, China). NOX4- small-interfering RNA (siRNA) and PFKFB3 siRNA were provided by GenePharma (Shang hai, China). Mouse PFKFB3 (NM_001177756) overexpressed plasmid was purchased from MiaoLing Plasmid Sharing Platform (Hubei, China). Other reagents are described below.
H2o2 assay kit
Manufactured by Beyotime
Sourced in China, United States
The H2O2 assay kit is a product designed to detect and quantify the presence of hydrogen peroxide (H2O2) in various samples. The kit provides a reliable and straightforward method to measure H2O2 concentrations, which is a crucial parameter in various scientific and industrial applications.
Automatically generated - may contain errors
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29 protocols using h2o2 assay kit
1
Apelin-13 Modulates Inflammatory Response
2
Hydrogen Peroxide Measurement in Fruit Flies
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Day 3 WT adult fruit flies were maintained on 1% agar containing 0.01 M C14-SPH. Ethanol was used as a control for 12 h before H2O2 levels were measured. Every 12 h, H2O2 was recorded till the 60th hour. H2O2 Assay Kit (Beyotime Biotechnology, Shanghai) was used to assay the H2O2 concentration. The H2O2 concentration determination was achieved by the oxidation of divalent iron ions. Their oxidation produced ferric ions and formed a purple product with xylenol orange in a particular solution. Tissue samples were homogenized at a ratio of 100 µl in lysis buffer from the kit per 5 mg of tissue. The lysis buffer could be substituted by phosphate-buffered saline (PBS, containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4) buffer. The samples were centrifuged at about 12,000 g for 5 min at 4°C and the supernatant was used for H2O2 measurement. All of the above operations need to be performed at 4°C or on ice. All tests were done in triplicate. H2O2 detection reagent was thawed on ice or on an ice water bath. The test was performed using 50 µl of samples and standard curve was set up by 50 µl of 1, 3, 10, 30, and 100 µM H2O2 standard solution. The absorbance at 560 nm was recorded on a uQuantMicroplate spectrophotometer (Biotek Instruments), and the concentration of H2O2 in the sample was calculated based on the standard curve.
3
Triacylglycerol and Oxidative Stress
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Triacylglycerol content in cells was detected by TG test kit (Mnzyme method, Solarbio Life Sciences, Beijing, China), H2O2 and malonaldehyde (MDA ) in cells were detected by H2O2 Assay Kit and Lipid Peroxidation MDA Assay Kit (Beyotime Biotechnology, Shanghai, China).
4
Quantifying Antioxidant Levels in Anthers
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The hydrogen peroxide (H2O2) content in anthers was measured using H2O2 assay kit (Beyotime, Shanghai, China) following the manufacturer’s instruction. The O2– radical content in anthers at each developmental stage was assayed using nitro-blue tetrazolium (NBT) staining according to a previous report (Xie et al., 2014 (link)).
The content of GSH was measured using GSH assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction. Carotenoid content was measured using plant carotenoid detection kit (Solarbio, Beijing, China) following the manufacturer’s instruction. The content of ascorbate peroxidase (APX) and GSH S-transferase (GST) was measured by APX and GST detection kit (GeRuiSi, Nanjing, China), respectively, according to the manufacturer’s instruction.
The content of GSH was measured using GSH assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction. Carotenoid content was measured using plant carotenoid detection kit (Solarbio, Beijing, China) following the manufacturer’s instruction. The content of ascorbate peroxidase (APX) and GSH S-transferase (GST) was measured by APX and GST detection kit (GeRuiSi, Nanjing, China), respectively, according to the manufacturer’s instruction.
5
Yeast Oxidative Stress Response Analysis
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Yeast cells in exponential phase were harvested and washed twice with cold sterile water followed by resuspension in lysis buffer containing acid-washed glass beads and 20 cycles of 10 s vortexing plus 20 s cooling [14 (link)]. After centrifugation at 12000 rpm for 15 min, the supernatants were collected and used for an enzyme activity assay and determination of intracellular H2O2 levels. The protein concentration was determined using the BCA protein Assay Kit (Sangon Biotech, Shanghai, China) following the manufacturer's instruction. The total superoxide dismutase (SOD) activity and the catalase activity were determined using a Total Cellular SOD Activity Kit (Dojindo Molecular Technologies, Rockville, MD, USA) and a Catalase Assay Kit (Beyotime Biotechnology, Shanghai, China), respectively. The intracellular H2O2 level was determined using an H2O2 Assay Kit (Beyotime Biotechnology, Shanghai, China).
6
Quantifying Intracellular H2O2 in Glioma Cells
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The content of intracellular H2O2 in treated glioma cells was analyzed with a H2O2 assay kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the manufacturer protocol. In brief, the cells were collected by centrifugation at 800 × g for 5 min and washed twice with PBS. The cell pellets and 10 mg xenograft glioma tissues were lysed in lysis buffer by repeated cycles of freezing and thawing under liquid nitrogen and centrifuged at 12,000 × g for 5 min. Then, 50 mL of supernatants and 100 mL of test solution were added into a tube, placed at room temperature for 30 min, and measured immediately with a spectrophotometer at a wavelength of 560 nm. Absorbance values were calibrated to a standard concentration curve to calculate the concentration of H2O2. The measurement was performed by a researcher who was blinded to group allocation and repeated for five times. Finally, the results were expressed as a ratio to the concentration of the control cells.
7
Measurement of Oxidative Stress Markers
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The probe MitoSOX red from Invitrogen company (Eugene, OR, USA), the probe DCFH-DA and the H2O2 assay kit from Beyotime Biotechnology (Nanjing, China) were used respectively to measure mitochondrial superoxide, intracellular ROS and H2O2 as described by us previously [15 (link)]. The results were expressed as a ratio to the control cells.
8
Quantifying Oxidative Stress Markers in Ischemic Stroke
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For in vivo studies, the mice were sacrificed at 3 h after cerebral ischemia/reperfusion. The brains were removed to obtain the intact ischemic lateral cortical tissue, and the tissue was homogenated on ice with a homogenizer. After centrifugation at 4°C, the supernatant was taken and the levels of MDA, lactic acid (LD), ATP and H2O2 were detected with MDA assay kit (S0131, Beyotime), LD assay kit (A019-2-1, NJJCBIO), ATP assay kit (S0026, Beyotime) and H2O2 assay kit (S0038, Beyotime) in accordance with the manufacturer’s recommendations. For in vitro studies, the ATP levels of primary cortical neurons were detected with ATP assay kit 3 h after OGD/R. The operating principle of ATP assay kit is based on the fact that firefly luciferase (also called luciferase) catalyzes luciferin to produce fluorescence and requires ATP to provide energy. When both firefly luciferase and luciferin are in excess, the production of fluorescence is proportional to the concentration of ATP within a certain concentration range. This allows highly sensitive detection of ATP concentration. The operating principle of hydrogen peroxide Assay Kit is that hydrogen peroxide can oxidize divalent iron ions to trivalent iron ions, and then form a purple product with xylenol orange in a specific solution.
9
Measuring H2O2 in Hyperuricemia Mice
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The H2O2 concentration was determined during UA degradation in vivo. The engineered EcN strains with oxygen-recycling genes or not were used to treat the hyperuricemia mice by either intragastric or intravenous administration, as described above. After the blood samples were taken, the H2O2 levels were detected by using a H2O2 assay kit (Beyotime S0038, Shanghai, China) according to the manufacturer’s instruction. Meanwhile, the UA concentration was determined with the UA meter.
10
Quantifying Tumor Hydrogen Peroxide Levels
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H2O2 levels were assessed using an H2O2 assay kit (Beyotime). Mouse tumor tissue (20 mg) was added to a 200 μL lysis buffer, homogenized, and centrifuged to obtain the supernatant. The supernatant (50 μL) and 100 μL of the test solution were combined in a tube at room temperature for 30 min, and the absorbance values at 560 nm were measured using a microplate reader (PerkinElmer). The absorbance values were calibrated to a standard concentration curve to calculate the concentration of H2O2.
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