The Human Interleukin 6 (IL-6) ELISA Kit (Invitrogen) and Human Tumor Necrosis Factor-α (TNF-α) ELISA Kit (Boster, Wuhan, China) were applied to evaluate the secretion levels of IL-6 and TNF-α in culture medium, respectively, as per the accompanying suggestion. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in hPDLCs after the indicated transfection or/and LPS exposure were gauged by the SOD Activity Colorimetric Assay Kit (Abcam) and MDA Colorimetric Assay Kit (Elabsience, Wuhan, China), respectively, based on the manufacturers’ recommendations.
Sod activity colorimetric assay kit
Manufactured by Abcam
Sourced in United States
The SOD activity colorimetric assay kit is a laboratory tool used to measure the activity of superoxide dismutase (SOD) enzymes in biological samples. The kit provides a colorimetric method for quantifying SOD activity, which is an important indicator of oxidative stress in cells and tissues.
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Lab products found in correlation
Lysozyme detection kit, by Merck Group (2 mentions)
Mpo colorimetric assay kit, by Merck Group (2 mentions)
Il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Tnf α elisa kit, by Boster Bio (1 mentions)
Bovine gamma globulin, by Bio-Rad (1 mentions)
Softmax pro, by Molecular Devices (1 mentions)
Mda assay kit, by Abcam (1 mentions)
Mpo colorimetric activity assay kit, by Merck Group (1 mentions)
8 ohdg elisa kit, by Abcam (1 mentions)
Elx800 plate reader, by Agilent Technologies (1 mentions)
Iron assay kit, by Abcam (1 mentions)
Lipid peroxidation mda assay kit, by Abcam (1 mentions)
In vitro ros rns assay kit, by Cell Biolabs (1 mentions)
Cat assay kit, by Merck Group (1 mentions)
11 protocols using sod activity colorimetric assay kit
1
Evaluating Cytokine and Oxidative Stress
2
Measurement of Antioxidant Enzyme Activities
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Glutathione peroxidase (GPx) activity was determined by measuring the rate of formation of oxidized glutathione form reduced GSH in the presence of H2O2, detected by the change in absorbance at 340 nm due to NADPH oxidation, as previously described58 (link).
Superoxide dismutase (SOD) activity was measured using the SOD activity colorimetric assay kit® (Abcam) according to the manufacturer’s protocol, in a microplate at 37 °C at 450 nm.
CI + III and CII + III activities was measured as previously described59 (link), 60 (link) with slight modifications. Reactions were adjusted to fit in 96-well microplate platform. NADH cytochrome c oxidoreductase and succinate cytochrome c reductase activities were acquired in SoftMax Pro (Molecular Devices) and expressed as the rate of reduced cytochrome c formation per min per mg of protein.
Cytochrome c oxidase activity was measured using a protocol based on a commercial kit (Sigma-Aldrich), which follows the decrease in absorbance at 550 nm due to the oxidation of reduced cytochrome c. Enzyme activity was calculated using the difference in reduced and oxidized cytochrome c molar absorptivity coefficients of 21.84 mM−1 × cm−1.
Enzyme activities are expressed relative to the total protein content, determined by Bradford (1976), using bovine gamma globulin (Bio-Rad) as standard.
Superoxide dismutase (SOD) activity was measured using the SOD activity colorimetric assay kit® (Abcam) according to the manufacturer’s protocol, in a microplate at 37 °C at 450 nm.
CI + III and CII + III activities was measured as previously described59 (link), 60 (link) with slight modifications. Reactions were adjusted to fit in 96-well microplate platform. NADH cytochrome c oxidoreductase and succinate cytochrome c reductase activities were acquired in SoftMax Pro (Molecular Devices) and expressed as the rate of reduced cytochrome c formation per min per mg of protein.
Cytochrome c oxidase activity was measured using a protocol based on a commercial kit (Sigma-Aldrich), which follows the decrease in absorbance at 550 nm due to the oxidation of reduced cytochrome c. Enzyme activity was calculated using the difference in reduced and oxidized cytochrome c molar absorptivity coefficients of 21.84 mM−1 × cm−1.
Enzyme activities are expressed relative to the total protein content, determined by Bradford (1976), using bovine gamma globulin (Bio-Rad) as standard.
3
Quantifying Antioxidant and Lipid Peroxidation
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The plasma levels of SOD and MDA were measured using the SOD activity colorimetric assay kit and the lipid peroxidation (MDA) assay kit according to the procedure supplied by the manufacturer (Abcam, Cambridge, MA, USA).
4
Serum Antioxidant Enzyme Profiling
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Superoxide dismutase (SOD), lysozyme, and myeloperoxidase (MPO) activities in serum were assessed using an SOD activity colorimetric assay kit (BioVision), a lysozyme detection kit (Sigma-Aldrich), and an MPO colorimetric assay kit (Sigma-Aldrich), respectively, according to the manufacturers’ instructions.
5
Antioxidant and Inflammatory Assays
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Superoxide dismutase (SOD) activity was expressed as the percentage of superoxide inhibition by using a SOD activity colorimetric assay kit (BioVision, USA) in accordance with the manufacturer’s instructions. The respiratory burst (RB) generated by phagocytes was measured via a nitro blue tetrazolium assay (Sigma-Aldrich, USA) as previously described22 . Myeloperoxidase (MPO) activity was analyzed with an MPO colorimetric activity assay kit (Sigma-Aldrich, USA). Antiprotease activity was expressed as the percentage of trypsin inhibition by using the method of Ellis23 . Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total glucose, total protein, triglyceride, and total cholesterols levels were measured with a BS-390 chemistry analyzer (Mindray Bio-Medical Electronics, China) at the Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, South Korea).
6
SOD Activity Colorimetric Assay
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SOD activity was determined by the SOD Activity Colorimetric Assay Kit (Biovision).
7
Assessing Antioxidant Capacity and DNA Oxidative Damage
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Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (K335-100, BioVision, Milpitas, CA, USA) was used for the examination of antioxidant capacity by assessing the water-soluble formazan dye upon reduction with superoxide anion to detect the inhibition activity of SOD. Briefly, the WST-1 working solution and the SOD enzyme working solution were added to the wells and mixed thoroughly. After incubation for 20 min, the inhibition activity of SOD was estimated using a microplate reader at 450 nm. The SOD activity (inhibition rate %) was calculated according to the manufacturer’s instructions.
8-hydroxy 2 deoxyguanosine (8-OHdG) ELISA Kit (ab201734, Abcam, Cambridge, UK) was utilized to measure the oxidative damage of DNA by oxidative stress according to the manufacturer’s protocol. Briefly, standards and samples were added into each well as instructed followed by incubation of HRP conjugated 8-OHdG antibody preparation for an hour. The enzymatic color reaction was developed after the TMB substrate solution was added and then the absorbance was measured at 450 nm.
8-hydroxy 2 deoxyguanosine (8-OHdG) ELISA Kit (ab201734, Abcam, Cambridge, UK) was utilized to measure the oxidative damage of DNA by oxidative stress according to the manufacturer’s protocol. Briefly, standards and samples were added into each well as instructed followed by incubation of HRP conjugated 8-OHdG antibody preparation for an hour. The enzymatic color reaction was developed after the TMB substrate solution was added and then the absorbance was measured at 450 nm.
8
Serum Biomarker Analysis in Immunology
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Serum superoxide dismutase (SOD), lysozyme, and myeloperoxidase (MPO) activity was analyzed using the SOD activity colorimetric assay kit (BioVision), the lysozyme detection kit (Sigma–Aldrich), and the MPO colorimetric assay kit (Sigma–Aldrich), respectively, according to the manufacturers’ instructions. Respiratory burst (RB) was analyzed using nitro blue tetrazolium (NBT) assay, as previously described (Hasan et al., 2018 (link)). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total glucose, and total cholesterols levels were measured using Mindray commercial kits and a Mindray BS-390 automatic biochemistry analyzer (Mindray Bio-Medical Electronics, China) at the Core-Facility Center, Dong-eui University (Busan, South Korea).
9
Colorimetric Assay for Superoxide Dismutase
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The activity of the antioxidant superoxide dismutase (SOD) was measured using a colorimetric SOD activity assay kit (Abcam, Pretoria, SA) as per the manufacturer’s instructions. Briefly, cell suspensions were prepared as described in section 1.6. The cells were lysed with 100 µl of 0.1 M trizma/hydrochloride (Tris/HCl), [comprising of 0.5% Triton X-100, 5 mM β-methylphenethylamine (β-ME) and 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF) at a pH of 7.4] and then transferred into 2 ml eppendorf tubes. Cell lysates were then centrifuged at 15,000 x g for 5 min at 4°C. Thereafter, 20 µl of the supernatants was pipetted into a new 96 well assay plate and to this, 200 µl of the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt (WST) working solution and 20 µl of the enzyme working solution was added to the supernatants. The plates were then incubated at 37 °C for 20 min. After incubation, SOD activity was measured on the Biotek® ELX 800 plate reader (Gen 5® software) at an absorbance rate of 450 nm.
10
Measuring Superoxide Dismutase Activity
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2 × 106 untreated HEI-OC1 cells or those treated with 80 µM t-BHP were collected and lysed with ice-cold cell lysis reagent (0.1 M Tris/HCl, pH 7.4, containing 0.5% Triton X-100, 5 mM β-ME, 0.1 mg/ml phenylmethylsulfonyl fluoride). Following centrifugation at 14,000 × g for 5 min, supernatants were collected and used for assays. SOD activity was measured using a colorimetric SOD activity assay kit (Abcam, United States) in 96-well plates. Briefly, 20 µl of the sample was mixed with WST working solution and enzyme working solution, and activities of SOD were calculated by measuring the absorbance at 450 nm following incubation at 33°C for 20 min. All experiments were performed in five replicates.
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