The levels of hydrogen peroxide (H2O2; another major antimicrobial component in honey) produced by the honey samples was determined using an Amplex Red hydrogen peroxide/peroxidase kit (Molecular Probes, Life Technologies Corp., Carlsbad, CA, USA) as previously described49 (link), and are included in Table 1 .
Amplex red hydrogen peroxide peroxidase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Amplex Red Hydrogen Peroxide/Peroxidase kit is a sensitive fluorometric assay for the detection and quantification of hydrogen peroxide (H2O2). The kit utilizes the Amplex Red reagent, which reacts with H2O2 in the presence of horseradish peroxidase (HRP) to produce the highly fluorescent compound resorufin. This reaction can be used to measure H2O2 levels in a variety of biological samples.
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12 protocols using amplex red hydrogen peroxide peroxidase kit
1
Quantifying Hydrogen Peroxide in Honey
2
Amplex Red Assay for H2O2 Quantification
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To measure H2O2 concentration of the worms, the Amplex red assay was performed as previously described [16 (link)] using the Amplex Red hydrogen peroxide/peroxidase kit (Invitrogen Molecular Probes, Eugene, OR) with the following modifications: L4 worms were exposed to a bacterial strain for 16 hours and transferred to 96 well plates with 30 worms in each well. A total of 80 mM diphenyleneiodinium chloride (DPI) (TCI, Tokyo) was added to some wells and allowed to incubate for 15 minutes prior to addition of Amplex Reagents. After 1 hour of incubation, fluorescence was measured at 540/590 nm excitation and emission, respectively.
3
Hydrogen Peroxide Quantification in C. elegans
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The Amplex Red hydrogen peroxide/peroxidase kit (Invitrogen Molecular Probes, Eugene, OR) was used to measure pathogen-stimulated hydrogen peroxide release by C. elegans as previously described (Chavez et al., 2007, 2009). Briefly, L4 worms were exposed to E. faecalis for 24 h, and the fluorescence of 10 worms per well was measured after 30 min incubation in the dark with Amplex Red/peroxidase solution (540/590 excitation and emission, respectively). The quantity of H2O2 was calculated using an etalon curve.
4
Measurement of Hydrogen Peroxide in MLE-12 Cells
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MLE-12 cells were cultured in phenol red free medium and then exposed to specific treatments. The cell culture supernatants were collected and H2O2 concentration was measured using Amplex Red Hydrogen Peroxide/Peroxidase kit (Invitrogen, United States), according to the manufacturer’s instruction.
5
Bacterial Hydrogen Peroxide Degradation
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Overnight cultures were subcultured at 1/100 in LB broth with appropriate antibiotics containing 1 mM H2O2 (VWR). Uninoculated LB broth containing 1 mM H2O2 was used as a control and was treated in parallel with other samples. Subcultures were incubated at 37°C with aeration, and aliquots for hydrogen peroxide detection were collected every hour. Bacteria were collected by centrifugation at maximum speed (Eppendorf 5415D) for 3 min. Cleared supernatants were used for hydrogen peroxide detection using an Amplex red hydrogen peroxide/peroxidase kit according to the manufacturer’s protocol (Invitrogen). Hydrogen peroxide concentrations correlated with production of resorufin, and fluorescence was measured at 530/585 nm. Results were expressed as percent hydrogen peroxide degradation calculated as [fluorescence530/585(tn)/fluorescence530/585 (t0)] * 100 over time. Each experiment was performed on at least three separate occasions. Statistical significance was determined using a Student’s t test.
6
Measuring H2O2 in Murine BAL Fluid
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BAL fluid from mice were collected and centrifuged at 10,000× g for 20 min at 4 °C, and H2O2 concentration was measured using Amplex Red Hydrogen Peroxide/Peroxidase kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.
7
Quantifying ROS Emission in Mitochondria
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ROS emission was quantified using the Amplex™ Red Hydrogen Peroxide/Peroxidase Kit (Invitrogen™, Carlsbad, CA, USA, #A22188) with slight modification to the manufacturer’s protocol. Liver mitochondria were diluted 1:4 in mannitol sucrose while skeletal muscle mitochondria were run undiluted. A quantity of 10 μL of each mitochondrial suspension was assayed in 190 μL of succinate buffer containing 500 mM KCl and 50 mM MgCl2 to measure peroxide production.
8
Quantifying Hydrogen Peroxide in EAC and BE Cells
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Hydrogen peroxide levels were measured following C-PAC treatment of EAC and BE cells using the Amplex Red Hydrogen Peroxide/Peroxidase kit according to the manufacturer’s recommendations (ThermoFisher Scientific, Waltham, MA). JHAD1 and OE19 EAC cells were plated at 10E3 and 15E3 cells/well, respectively in 96-well black walled, clear bottom plates (Greiner Bio One, Monroe, NC). CP-C BE cells were plated at 8E3 cells/well in the same plates. Cells adhered for 26–30 h, washed once with PBS and treated for 6 h with vehicle, C-PAC [50–100 μg/mL], the inducer 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ; 20 μM final concentration; Sigma Aldrich, St. Louis, MO) and the inhibitor diethylene triamine pentaacetic acid (DTPA; 100 μM final concentration; Sigma Aldrich, St. Louis, MO). After 6 h, the supernatant was transferred to a new plate and the cells were washed. The cells, the supernatant from the cells and the reaction-no cells in medium were assayed independently for hydrogen peroxide 30 min after addition of the Amplex Red substrate. Fluorescence was measured using the SpectraMax® i3 with excitation/emission wavelengths of 545–590 nm. A minimum of 4 wells were analyzed for each treatment and the data expressed as mean relative fluorescence units ± SEM.
9
Quantifying Mitochondrial Hydrogen Peroxide
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In addition to their critical role in ATP synthesis, mitochondria are also major source of ROS in cells. It has been suggested that 2% of the oxygen consumed by mitochondria is converted to superoxide. In turn, superoxide is converted by manganese superoxide dismutase to H2O2. We used Amplex red hydrogen peroxide/peroxidase kit (Thermo Fischer Scientific) to detect H2O2 in 0.3 cm2 well of the plate with 100 µl of total volume61 (link). The method uses 10-acetyl-3,7-dihydroxyphenoxazine (10 µM) in combination with HRP. H2O2 released from cell, reacts with 10-acetyl-3,7-dihydroxyphenoxazine in the presence of peroxidase and generates resofurin (red-fluorescent oxidation product), which is detected at excitation and emission of 571 nm and 585 nm, respectively. The concentration of H2O2 was determined with the help of a standard curve prepared during experiment.
10
Measuring Hydrogen Peroxide in CFCS
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Hydrogen peroxide levels present in CFCS were measured using the Amplex Red Hydrogen Peroxide/Peroxidase kit according to the manufacturer’s recommendations (Thermo Fisher Scientific, Waltham, MA, USA). After preparing the kit stock solutions, aliquots (50 μL) of the standard curve samples, controls, and experimental samples were added to individual wells on a microplate. The Amplex Red reagent/HRP working solution (50 μL) was added to the wells previously plotted. After incubation (30 min, room temperature - 25°C, protected from light), the absorbance was measured in a microplate reader (550 nm) to construct the standard curve and measure the H2O2 concentration (μM) of the CFCS.
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