Direct binding duplex ELISAs measured immunoreactivity to RCAN1 and IDE, followed by large acidic ribosomal protein (RPLPO) [107] (link) to normalize expression of target proteins. RCAN1 and IDE were detected with horseradish peroxidase–conjugated secondary antibody and Amplex UltraRed (Invitrogen, Carlsbad, CA), and RPLPO was subsequently detected with biotinylated anti-RPLPO (Proteintech Group Inc, Chicago, IL) and streptavidin-conjugated alkaline phosphatase with 4-methylumbelliferyl phosphate (4-MUP). Fluorescence intensities (Amplex Red: Ex565/Em595; 4-MUP: Ex360/Em450) were measured in a SpectraMax M5 (Molecular Devices, Sunnyvale, CA). The calculated specific protein-to-RPLPO ratios were used for intergroup statistical comparisons.
Amplex red
Manufactured by Molecular Devices
Sourced in United States
Amplex Red is a fluorogenic substrate used for the detection and quantification of hydrogen peroxide (H2O2) in biological samples. It undergoes a reaction catalyzed by the enzyme horseradish peroxidase (HRP) to produce a highly fluorescent product, resorufin, which can be measured using a fluorescence spectrophotometer.
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Lab products found in correlation
Amplex red, by Thermo Fisher Scientific (9 mentions)
Flexstation 3, by Molecular Devices (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Amplex ultrared, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti rplpo, by Proteintech (1 mentions)
Opteia elisa kit, by BD (1 mentions)
Spectramax m2 microplate reader, by Molecular Devices (1 mentions)
Flexstation ii384, by Molecular Devices (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Spectramax m2 m2e microplate reader, by Molecular Devices (1 mentions)
H2dcfda, by Molecular Devices (1 mentions)
Spectramax plate reader, by Molecular Devices (1 mentions)
Paraquat, by Merck Group (1 mentions)
Spectramax gemini xs, by Molecular Devices (1 mentions)
10 protocols using amplex red
1
Protein Expression Quantification by ELISA
2
Mitochondrial Hydrogen Peroxide Production
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Hydrogen peroxide production in isolated mitochondrial subpopulations was determined using the oxidation of the fluorogenic indicator Amplex Red (Invitrogen) in the presence of horseradish peroxidase as previously described [21] (link). The concentrations of horseradish peroxidase and Amplex Red in the incubation were 0.1 unit/mL and 50 μM, respectively, and detection of fluorescence was assessed on a on a Molecular Devices Flex Station 3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with 530-nm excitation and 590-nm emission wavelengths. Standard curves were obtained by adding known amounts of H2O2 to the assay medium in the presence of the substrates Amplex Red and horseradish peroxidase. H2O2 production was initiated in mitochondria using glutamate + malate (G + M) and succinate + rotenone (S + R) as substrates.
3
Cytokine and ROS Production in Splenic Macrophages Infected with T. cruzi
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Splenic Mo/Mφ were in vitro incubated for 24 h in presence or absence of T. cruzi, SRT1720 and FAK inhibitor. Culture supernatants were analyzed by using optEIA™ ELISA kits (Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions to examine the cytokines (TNF-α, IL-6, and IL-10) release. Standard curves were prepared using the recombinant cytokines.
For the measurement of reactive oxygen species (ROS) release, culture supernatants (50 μL/well) were added in triplicate to 96-well black flat-bottomed plates. Samples were mixed with 50 μL of 100 μM 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Molecular Probes, Eugene, OR, USA) and 50 μL of 0.1 U/mL horseradish peroxidase. The H2O2-dependent oxidation of Amplex Red to fluorescent resorufin (Ex563nm/Em587nm) was recorded by using a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA) [17 (link)].
For the measurement of reactive oxygen species (ROS) release, culture supernatants (50 μL/well) were added in triplicate to 96-well black flat-bottomed plates. Samples were mixed with 50 μL of 100 μM 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Molecular Probes, Eugene, OR, USA) and 50 μL of 0.1 U/mL horseradish peroxidase. The H2O2-dependent oxidation of Amplex Red to fluorescent resorufin (Ex563nm/Em587nm) was recorded by using a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA) [17 (link)].
4
Mitochondrial Hydrogen Peroxide Production
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Hydrogen peroxide production in isolated mitochondrial subpopulations IFM and SSM was determined using the oxidation of the fluorogenic indicator Amplex Red (Invitrogen) in the presence of horseradish peroxidase as previously described (28 (link)). The concentrations of horseradish peroxidase and Amplex Red in the incubation were 0.1 unit/mL and 50μM, respectively, and detection of fluorescence was assessed on a Molecular Devices Flex Station 3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with 530-nm excitation and 590-nm emission wavelengths. Standard curves were obtained by adding known amounts of H2O2 to the assay medium in the presence of the substrates Amplex Red and horseradish peroxidase. H2O2 production was initiated in mitochondria using glutamate + malate (G + M) and succinate + rotenone (S + R) as substrates.
5
Measuring Cellular Oxidative Stress
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To assess the effects of palmitate and/or Seps1 knockdown on cellular ROS, myotubes were incubated with 10 μmol/L 5‐(and‐6)‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate (DCFDA; Thermo Fisher Scientific). DCFDA oxidation was detected using a FlexStationII384 fluorometer (Molecular Devices; excitation = 485 nm and emission = 538 nm). H2O2 (25 μmol/L) was used as a positive control.
Amplex® Red (10‐acetyl‐3,7‐dihydroxyphenoxazine; 50 μmol/L; Thermo Fisher Scientific) and 0.1 U/mL of horseradish peroxidase (HRP) were used to measure H2O2 production in muscle cells following Seps1 knockdown and/or palmitate treatment. Resorufin, the reaction product of Amplex® Red oxidation, was detected using a FlexStation II384 fluorometer (Molecular Devices; excitation = 570 nm and emission = 585 nm). Glucose oxidase (25 mU/ml) was used as a positive control. The increase in absorbance (FU) was normalized to the protein content of each well and presented as a fold‐change increase in H2O2.
Reduced (GSH) and oxidized (GSSG) glutathione levels were measured, as previously described (Conlan et al.2010 ), to determine the GSH:GSSG ratio in myotubes following 24 h of palmitate treatment or in myoblasts following Seps1 gene suppression.
Amplex® Red (10‐acetyl‐3,7‐dihydroxyphenoxazine; 50 μmol/L; Thermo Fisher Scientific) and 0.1 U/mL of horseradish peroxidase (HRP) were used to measure H2O2 production in muscle cells following Seps1 knockdown and/or palmitate treatment. Resorufin, the reaction product of Amplex® Red oxidation, was detected using a FlexStation II384 fluorometer (Molecular Devices; excitation = 570 nm and emission = 585 nm). Glucose oxidase (25 mU/ml) was used as a positive control. The increase in absorbance (FU) was normalized to the protein content of each well and presented as a fold‐change increase in H2O2.
Reduced (GSH) and oxidized (GSSG) glutathione levels were measured, as previously described (Conlan et al.
6
Mitochondrial ROS Measurement Using Amplex Red
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Amplex Red (Life Technologies, Carlsbad, CA), a dye that reacts with H2O2 to produce the highly fluorescent molecule, resorufin, was used for measurement of ROS levels in isolated mitochondria. Mitochondria were incubated with 100 μM Amplex Red for 15 min, and the fluorescence intensity was quantified using a Spectramax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation of 460 nm and emission of 490 nm.
7
Oxidative Stress Biomarkers Analysis
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The protein carbonyls in the tissue homogenates were measured by colorimetric protein carbonyl assay (Cayman Chemicals, cat# 10005020), according to the instructions provided by the manufacturer. Protein carbonyl content is expressed in nmol/mg of protein. ROS release was measured by Amplex red assay. Briefly, homogenized samples of heart tissue (50 µg protein) were added to black flat-bottom plates. The reaction was initiated by the addition of 50 µL each of 100 μM Amplex red (Invitrogen) and 0.3 U/mL HRP. The HRP catalyzed Amplex red oxidation by H2O2, resulting in fluorescent resorufin formation, was monitored at Ex563 nm/Em587 nm (standard curve: 50 nM–5 M H2O2) using a Spectra Max M2 microplate reader (Molecular Devices, San Jose, CA, USA). A thiobarbituric acid-reactive substances (TBARS) assay was used to measure MDA-TBA adduct formation using the Cayman TBARS assay kit (cat# 700870). The MDA-TBA adduct was measured colorimetrically at 530–540 nm using a microplate reader (BioTek, Winooski, VT, USA). The MDA concentration is expressed in nM/mg protein.
8
Quantifying Intracellular H2O2 and ROS Levels
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Intracellular levels of H2O2 or ROS were assessed using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Molecular Probes) to evaluate H2O2 or carboxy-2′ 7’ dihydrodichlorofluorescein diacetate (H2DCFDA, Molecular Probes) to assess total ROS. Cells were incubated in a phenol-free medium in the presence of 50 μM Amplex Red and 0.1 U/mL horseradish peroxidase or 10 μM H2DCFDA under specific treatment conditions. Moreover, cells were incubated with 1 μM of ROS-insensitive probe carboxy-DCFDA (Invitrogen) instead of H2DCFDA for the negative control group. Fluorescence was measured using a SpectraMax M2/M2e Microplate Reader (Molecular Devices) with excitation at 530 nm and emission at 590 nm for Amplex Red, or excitation at 485 nm and emission at 520 nm for H2DCFDA.
9
LOX Enzyme Activity Measurement Protocol
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LOX enzyme activity was measured by Palamakumbura's modified protocol [18, 19] . Active LOX in the sample reacts with the pseudosubstrate cadaverine and the H 2 O 2 generated in the reaction is able to oxidise the Amplex Red to resorfurin, a fluorescence product. The assay reaction mixture consisted of 50 mm sodium borate (pH 8.2), 50 µm Amplex Red (N-acetyl-3,7 dihydroxyphenoxazine, Molecular Probes, Invitrogen), 0.1 U/mL horseradish peroxidase, and a naturally occurring diamine substrate, 10 mm 1,5-diaminopentane, cadaverine (Sigma, St. Louis, USA). Briefly, 5 µL of AH was added to the final reaction mixture. Resorfurin is the final product of Amplex Red assay, whose fluorescence is measured in kinetic mode at 37°C at 563 nm excitation, and the emission was read at 587 nm using a SpectraMax plate reader (Molecular Devices, USA). A standard graph was obtained using H 2 O 2 (0.2-1.0 µm). The relative fluorescence unit obtained in test samples was calculated based on the standard and was expressed as unit activity (micromoles H 2 O 2 /minute). We measured only total LOX activity and did not perform specific assays for the LOX isoforms.
10
Mitochondrial ROS Production Assay
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To measure ROS production, mitochondria were isolated from untreated E6-1 or HT-29 cells. Cells were washed twice in cold PBS, resuspended in hypotonic buffer (1 mM EDTA, 5 mM Tris-HCl, 100 mM mannitol, 70 mM sucrose, 10 µM Leu-pep, 10 µM Pep-A, and 100 µM PMSF) and immediately homogenized, and centrifuged at 600× g for 5 minutes at 4°C. The supernatant was centrifuged at 15,000× g for 15 minutes at 4°C. The resulting cytosolic supernatant was discarded and the mitochondrial pellet was resuspended in cold hypotonic buffer. To measure ROS levels, Amplex Red (Molecular Probes, Eugene, OR, USA. Cat. No.A12222) was used. The isolated mitochondria were resuspended in cold hypotonic buffer and 150 µg of protein was added to wells of a 96-well opaque plate. Cells were treated with ethanolic or aqueous NLE and as a positive control, 250 µM paraquat (Sigma Aldrich, Mississauga, ON, CA. Cat. No.75365-730) was used for treatment. Amplex Red reagent was added to each well for a final concentration of 50 µM; horseradish peroxidase (HRP) was added in the ratio of 6 units per 200 µL. Fluorescence readings were taken every 5 minutes for a total time of 5 hours at Ex. 560 nm and Em. 590 nm on a spectrofluorometer (SpectraMax Gemini XS, Molecular Devices, Sunnyvale, CA). The readings were analyzed using GraphPad Prism 5.0 software and expressed as relative fluorescence units (RFU).
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