H2O2 levels were monitored using the Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences) according to the manufacturer’s protocol. Cells from 3 ml of culture were harvested and resuspended in lysis buffer (20 mM phosphate buffer, 5 mM EDTA, 0.2 mM PMSF, pH 7.2). Cells were broken using glass beads (0.5 mm diameter) by vortexing five times for 1 min with intervals of 1 min on ice. Cell debris was pelleted and the supernatants used for the test. The conversion of red peroxidase substrate to resorufin was determined measuring OD576. Data were expressed as fold increase with respect to the empty vector strain level.
Red hydrogen peroxide assay kit
Manufactured by Enzo Life Sciences
Sourced in United States
The Red Hydrogen Peroxide Assay Kit is a laboratory tool used to quantify the concentration of hydrogen peroxide (H2O2) in a sample. It utilizes a colorimetric detection method that produces a red-colored product proportional to the amount of H2O2 present.
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Lab products found in correlation
Superoxide dismutase assay kit, by Cayman Chemical (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Paradigm detection platform, by Molecular Devices (1 mentions)
Zircoprep mini, by Nippon Genetics (1 mentions)
Victor3 plate reader, by PerkinElmer (1 mentions)
Lambda 25 uv vis spectrophotometer, by PerkinElmer (1 mentions)
Quantichrom heme assay kit, by BioAssay Systems (1 mentions)
Pierce microplate bca protein assay kit reducing agent compatible kit, by Thermo Fisher Scientific (1 mentions)
Tristar lb 941, by Berthold Technologies (1 mentions)
Zircoprep mini kit, by Nippon Genetics (1 mentions)
9 protocols using red hydrogen peroxide assay kit
1
Quantifying Cellular Hydrogen Peroxide
2
Quantifying Peroxide Levels in Organic Compounds
3
Quantifying Hydrogen Peroxide Levels
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HaCaT keratinocytes were seeded in a black 96 well plate overnight until confluence. The plate was washed using PBS (−), and cells were treated with ROS scavengers, WEB2086, or diphenyleneiodonium chloride (DPI) prior to 1 mM EP stimulation similar to previous assays. The same method was used to measure hydrogen peroxide concentration in media only with EP stimulation. Following incubation, Red Hydrogen Peroxide assay kit (Enzo Life Sciences, Farmingdale, NY) was used in accordance to the manufacturer’s instructions, and fluorescence intensity of the standards was measured using a microplate reader at a wavelength of Ex570/Em590 nm. The standards curve equation was then used to calculate the hydrogen peroxide concentration of the samples.
4
Quantifying Antioxidant Defenses in Oral Bacteria
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S. mutans ATCC25175, S. oralis ATCC6249, and P. gingivalis ATCC33277, which are indigenous to the oral cavity, were inoculated with the same broth containing SPE at various dilutions. Briefly, inoculated samples were incubated aerobically or anaerobically at 37 °C for 24 h. Bacterial cells were harvested through centrifugation at 5000× g for 10 min under 4 °C chamber conditions and washed twice with Phosphate buffered saline (PBS). Cells (approximately 1.0 × 108) were suspended in 150 μL solution containing 50 mM Tris-HCl buffer (pH 7.6), 1 mM EDTA, and 0.5% triton-X 100. Subsequently, the suspension was placed in ZircoPrep Mini (Nippon Genetics Co. Ltd., Tokyo, Japan) and agitated for 10 min in a Cell disruptor (µT-12, TAITEC, Tokyo, Japan). After sample centrifugation, supernatant was used.
Superoxide Dismutase Assay Kit (Cayman Chemical Company Inc., Ann Arbor, MI, USA) was used to establish SOD amounts in tested bacterial cells, whereas Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) was used to establish bacterial hydrogen peroxide amounts following our earlier work [49 (link)]. Both kits were performed according to the manufacturer’s recommendation.
Superoxide Dismutase Assay Kit (Cayman Chemical Company Inc., Ann Arbor, MI, USA) was used to establish SOD amounts in tested bacterial cells, whereas Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) was used to establish bacterial hydrogen peroxide amounts following our earlier work [49 (link)]. Both kits were performed according to the manufacturer’s recommendation.
5
Hydrogen Peroxide Quantification Assay
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Hydrogen peroxide levels were determined using Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences) according to manufacturer's protocol. 10 mL of cells induced for 16 h were harvested and resuspended in lysis buffer (20 mM phosphate buffer, 5 mM EDTA, 0.2 mM PMSF, pH 7.2). Cells were broken using glass beads (0.5 mm diameter) by vortexing eight times for 1 min with intervals of 1 min on ice. Supernatant was removed to new tubes and the concentration of total protein was determined by Bradford method. The conversion of red peroxidase substrate was determined by monitoring the fluorescence increase with an excitation at 531 and an emission filter at 595 nm in a Victor 3 Plate Reader (Perkin-Elmer).
6
Characterization of Bdr Enzyme Kinetics
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Bdr was reduced by addition of 1 mM DTT and incubated on ice for 5 min. Bdr was buffer exchanged twice using buffer exchange micro-spin columns (Bio-RAD) that were pre-washed three times with 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA and 10% glycerol. Protein concentration was determined using the Bradford assay with BSA as standard. Bdr (typically at 5 μM) consumption of NADPH was assessed in 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA and 10% glycerol buffer by monitoring OD340 using a 1 cm pathlength quartz cuvette in a PerkinElmer Lambda 25 UV/VIS Spectrophotometer. Reactions were started by rapid mixing of 0.1 mM NADPH and OD340 was measured at different time intervals. H2O2 was measured using Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences) according to the manufacturer's instructions. BSH levels were measured using 5,5′-dithiobis-(2-nitrobenzoic acid) (DNTB).
7
Quantifying Inflammation and Stress Signals
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Secreted inflammation-related signals quantified include IL-8 and IL-6 cytokines, whereas secreted stress-related signals measured include total heme and hydrogen peroxide (H2O2). Additionally, caspase-1 (CASP1) and caspase-3 (CASP3) were likewise quantified to elucidate whether detected inflammation- and stress-related signals resulted in cell death. Pierce® Microplate BCA Protein Assay Kit-Reducing Agent Compatible Kit (Thermo Scientific, Carlsbad, CA, USA) was used to standardize cell-free supernatant prior to downstream analyses. Secreted IL-8 and IL-6 amounts in the cell-free supernatants were measured using commercially available IL-8 and IL-6 ELISA kits (R&D Systems, Minneapolis, MN, USA). QuantiChromTM Heme Assay Kit (BioAssay Systems, Hayward, CA, USA) was used to measure secreted heme (free heme and heme-proteins). Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences, Plymouth Meeting, Pennsylvania, PA, USA) was used to measure secreted H2O2. Secreted CASP1 and CASP3 were quantified usingCaspase-1/ICE and Caspase-3/CPP32 Colorimetric Assay Kits (Biovision, Milpitas, CA, USA), respectively. All kits were used following the manufacturer’s recommendations.
8
Quantifying Endogenous H2O2 and Peroxidase in Arabidopsis
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Measurements of endogenous concentrations of H2O2 and peroxidase activities in Arabidopsis leaf tissues were measured by using the Red Hydrogen Peroxide Assay Kit (Enzo Life Science) according to the manufacturer’s instructions. The harvested samples (100 mg) were ground in liquid N2 and suspended in 200 μl of 20 mM sodium phosphate buffer (pH 7.4). The mixture was centrifuged at 9,500g for 10 min at 4°C, and the supernatant was used for the subsequent assays.
9
NTAAP Treatment Effects on Bacterial Antioxidants
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A Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) and a Superoxide Dismutase Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) were used to measure bacterial H 2 O 2 and SOD levels, respectively, in accordance with the manufacturer's instructions. In brief, the bacterial cell suspensions described above were treated with the NTAAP device for 5 min (S. mutans), 1 min (P. gingivalis), and 7 min (E. faecalis). The treatment distance was fixed at 1 mm. After NTAAP treatment, the bacteria were lysed with a ZircoPrep Mini Kit (Nippon Genetics Co., Ltd., Tokyo, Japan). For H 2 O 2 quantification, the H 2 O 2 detection solution was mixed with the bacterial lysate and incubated at room temperature for 30 min. Sample densities were measured using a spectrometer (TriStar LB 941, Berthold Technologies, Bad Wildbad, Germany) at 570 nm. For SOD quantification, the SOD detection solution was mixed with the bacterial lysate and incubated in a plate shaker for 20 min at room temperature. Absorbance was then measured at 405 nm using a spectrometer. The protein content of the bacterial lysate was also determined quantitatively using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and both the H 2 O 2 and the SOD levels were normalized to the protein content.
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