The production of H2O2 in the light-driven assays was measured using Ampliflu™ Red (Sigma) according to Singh et al. [45 (link)]. All reactions were performed in black 96-well Nunc™ optical plates (Thermo Scientific). The assay was sampled every 10 min and 10 µL samples were mixed with 2 µL Ampliflu™ Red (5 mM stock), 15 µL Horseradish Peroxidase from Sigma (300 U mL−1), 2 µL Ethylenediaminetetraacetic acid (EDTA) (10 mM stock), and 171 µL 50 mM K2PO4 (pH 6.0). Measurements were made on Biotek Synergy™ microplate reader preheated to 37 °C and analyzed with Gen5™ Data Analysis Software. Excitation was set to 557 nm and emission was measured at 583 nm with three replicate reads per reaction. A standard curve was prepared from ≥ 30% H2O2 for trace analysis from Sigma Aldrich. The assays were composed of varying conditions included light intensity (0, 50, 100, 200, 500 µmol m−2 s−1) and ascorbic acid (0, 250, 500, 1000 µM). All experiments were performed in triplicates and data shown are the averages with the standard error of the mean (SEM).
Ampliflu red
Manufactured by Merck Group
Sourced in Germany
Ampliflu™ Red is a fluorescent dye used in nucleic acid detection and quantification applications. It binds to double-stranded DNA, producing a red fluorescent signal that can be detected and measured using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by Merck Group (3 mentions)
Synergy h1, by Agilent Technologies (3 mentions)
Spectramax microplate reader, by Molecular Devices (2 mentions)
Tyramine hydrochloride, by Merck Group (2 mentions)
Selegiline, by Merck Group (2 mentions)
Hmao a, by Merck Group (2 mentions)
Hmao b, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (2 mentions)
Infinite 200, by Tecan (1 mentions)
Moclobemide, by Merck Group (1 mentions)
Victor x4 2030 multilabel reader, by PerkinElmer (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Arachidonic acid, by Cayman Chemical (1 mentions)
Sc 560, by Cayman Chemical (1 mentions)
Dup 697, by Cayman Chemical (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
Sodium azide, by Thermo Fisher Scientific (1 mentions)
Sodium fluoride, by Thermo Fisher Scientific (1 mentions)
Phloretin, by Merck Group (1 mentions)
27 protocols using ampliflu red
1
Quantifying Light-Driven H2O2 Production
2
Mitochondrial ROS Production Assay
3
Quantification of Hydrogen Peroxide Production
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Hydrogen peroxide (H2O2) production was measured using Ampliflu Red (Sigma-Aldrich) reagent. ECs were seeded on 96-well plate to reach 90% confluence. To assess total H2O2, the cell culture medium was substituted with PBS + 2% w/v BSA + 0.7 mM MgCl2 and 0.7 mM CaCl2 containing 40 µM Ampliflu Red reagent, 1 µg/mL HRP, 5 µg/mL SOD and 100 µM NaN3 in a final volume of 100 µL. After 5 min of preincubation with TNF-α (100 ng/mL) or Histamine 10−5 M, the readings were taken at 576 nm with Infinite200 (TECAN). The cells were then lysed for the quantification of the total protein concentration by Bradford assay [24 (link)].
4
Fluorometric Measurement of Adipocyte Oxidative Capacity
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Fat pads or adipocytes were homogenized in PBS, supplemented with PI and PhoI, and centrifuged at 4°C and 2,000 g for 10 min. Supernatants were collected, and the fluorescence was measured at 37°C after the addition of 100 μM NADPH (N5130; Sigma-Aldrich), 100 μM AmpliFlu Red (90101; Sigma-Aldrich), and 1 U/ml horseradish peroxidase (P8250; Sigma-Aldrich). The final relative fluorescence unit signal was normalized according to the protein concentration, which was determined by the absorbance at 280 nm. The fluorescence was measured under the following settings: excitation, 535 nm; emission, 595 nm; gain, 50.
5
Measuring Mitochondrial ROS Production
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To estimate mtROS production, H2O2 levels were measured in freshly isolated mitochondria (50 μg) at 37°C by the SpectraMax Microplate Reader (Molecular Devices). The mitochondria were incubated at 37°C in incubation buffer (in mM): 125 KCl, 20 Tris-base, 2 KH2PO4, 1 MgCl2, 1 EGTA, 5 α-ketoglutarate, 5 L-malate, pH 7.1. Production of H2O2 was measured in the medium containing 0.1 mM Ampliflu™ Red (Sigma), 50 mM sodium phosphate pH 7.4, 0.2 U/mL HRP. External Ca2+ was added with 5-min intervals to increase a total (accumulative) concentration of the ion in the incubation medium up to 100, 200, 300, and 800 μM. The fluorescence intensity of Ampliflu™ Red was determined at the excitation/emission of 560 nm/590 nm. Results were expressed as pmol of H2O2 per min in 1 mg of protein.
6
Detecting LPMO-Catalyzed Hydrogen Peroxide Generation
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Generation of hydrogen peroxide by LPMOs was detected using the Amplex Red assay (29 (link)), which employs Ampliflu Red (10-acetyl-3,7-dihydroxyphenoxazine; 90101; Sigma-Aldrich) and horseradish peroxidase (HRP; P8125; Sigma-Aldrich). A 200-μl reaction mixture contained 0.05 mM Ampliflu Red, 3.55 U ml−1 HRP, 0.05 mM ascorbic acid (AscA), and 0.4 to 3.3 μM enzyme in 50 mM sodium citrate buffer, pH 6.0. The reaction was measured at 30°C for 30 min on a FLUOstar Omega microplate reader by recording fluorescence (excitation, 544 nm; emission, 600 nm) every 60 s. A standard curve was prepared with H2O2 (0.25 to 5 μM).
7
Enzymatic Inhibition Profiling of Novel Compounds
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To investigate the MAO-A and MAO-B enzyme inhibition profiles of the synthesized compounds, the fluorometric enzyme inhibition assay was carried out, as previously defined by us [30 (link),38 (link),39 (link),40 (link)]. Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine) was used as a fluorescence reagent in this assay. All reagents and enzymes (Ampliflu™ Red, peroxidase from horseradish, hMAO-A, hMAO-B, H2O2, tyramine hydrochloride, moclobemide and selegiline) were supplied by Sigma-Aldrich (Steinheim, Germany).
8
Catalase Activity Assay in Fluid Fraction
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The HPSC was evaluated in FF samples following adaptation of Aebi’s method [48 (link), 49 (link)]. Briefly, the FF samples were diluted (1:75) in Tris-HCL 0.1 mol/L pH 7.5 buffer. Then, 250 µL of 40 µM H2O2 solution was added to 250 µL of diluted sample or the catalase standard curve standards, and all samples were incubated at 37°C for 30 minutes. Then, 50 µL of samples and standards were placed on 96-well microplates in duplicate and 100 µL of a solution containing 0.4 U/mL horseradish peroxidase (HRP), Tris-HCL 0.1 mol/L pH 7.5 buffer, and Ampliflu Red (Sigma-Aldrich). In this reaction, HRP uses Ampliflu Red as an electron donor during the reduction of H2O2 to water, and fluorescent resorufin is formed. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, and it was measured with a spectrofluorometer (Victor X42030 Multilabel Reader, PerkinElmer) at 37°C. The results were expressed as arbitrary units of intensity (A.U.). The samples were measured in 3 different assays. All experiments were performed in duplicate.
9
Measuring Oxidative Stress Resistance in Rotifers
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We assessed the age-specific levels of oxidative stress resistance due to enzymatic processes in A. vaga by measuring the combined ability of catalase and peroxidase to remove H2O2 from solution. The ability of extracts to remove H2O2 from solution was assayed using an amplex red catalase assay protocol derived from that used with the Invitrogen kit (A22180) but substituting Ampliflu Red (Sigma) and laboratory stock reagents (i.e., anhydrous dimethylsulfoxide, horseradish peroxidase, 3% H2O2, 0.5 M Tris–HCl at pH 7.5, and catalase). Standard curves for catalase activity were generated using purified enzyme (Sigma) serially diluted in 1X reaction buffer (Tris–HCl). Twenty-five microliter of 40 µM H2O2 was added to 25 µL of each standard or sample in a 96-well plate and incubated for 30 min at room temperature, after which 50 µL of amplex red working solution was added and incubated for an additional 30 min at room temperature. Fluorescence emission was assayed at 590 nm with excitation at 530 nm and optimal gain on a Tecan Infinite M200 Pro plate reader.
10
Western Blot Analysis of RUNX3 Protein
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Preparation of whole-tissue extracts of the tumour specimens, SDS-PAGE, and protein transfer to nitrocellulose membrane procedures was done as previously described [32 (link)]. Primary rabbit antibody against RUNX3 (Antibodies-Online, cat no. ABIN739370) diluted 1:500 in 5% nonfat milk in PBS was used for RUNX3 protein detection applying 2-hour incubation on a platform shaker at room temperature. After washing in PBS buffer supplemented with 0.5% of Tween-20, membranes were incubated with the anti-rabbit secondary antibody conjugated with horseradish peroxidase (Life Technologies, cat no. 656120, dilution 1:2000) for 1-hour at room temperature. Signals were visualized using enhanced chemiluminescence reagent Ampliflu™ Red (Sigma-Aldrich, cat no. 90101) and recorded using gel visualization system GelDoc-It®2 imager (Analytik Jena AG). Detection assay of input control, ACTB, on the same membranes after mild stripping and reprobing was performed as previously described [32 (link)]. Values of RUNX3 and ACTB signals were calculated applying image analysis program ImageJ version 1.47 (National Institutes of Health, USA).
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