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Oxiselect total antioxidant capacity assay kit

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The OxiSelect Total Antioxidant Capacity Assay Kit is a quantitative colorimetric assay that measures the total antioxidant capacity of biological samples. The kit utilizes a copper (II) reagent that is reduced by antioxidants in the sample, resulting in a colorimetric change that can be measured spectrophotometrically.

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47 protocols using oxiselect total antioxidant capacity assay kit

1

Evaluating Antioxidant Status and Body Composition

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At baseline, the samples for the standard analysis and HS-Omega-3 Index were collected during the fasting state. A 20 mL blood sample was taken by venipuncture, processed and stored for analysis of the HS-Omega-3 Index and standard health parameters at baseline, the HS-Omega-3 Index two months after study start and at the end of the supplementation period, as well as the total antioxidant capacity (TAC) and malonyl dialdehyde (MDA) concentrations before and after the training session.
The TAC in plasma samples was measured using the OxiSelect™ total antioxidant capacity assay kit (STA-360, Cell Biolabs Inc., San Diego, CA, USA) following the manufacturer’s instructions. High values in the TAC assay reflect a high antioxidant capacity, i.e., greater protection. Uric acid equivalent was used to calculate µM copper-reducing equivalent values. The level of MDA was measured colorimetrically using the OxiSelect™ TBARS assay kit (MDA Quantitation, STA-330, Cell Biolabs Inc., San Diego, CA, USA). MDA quantitation results are expressed as µmol/L (µM) concentration using the MDA standard curve obtained during the processing of the plasma samples.
Body composition was assessed by kinanthropometry following international standards for anthropometric assessments applying the Yuhasz formula modified by Faulkner [59 ,60 ].
Drobnic F., Storsve A.B., Burri L., Ding Y., Banquells M., Riera J., Björk P., Ferrer-Roca V, & Domingo J.C. (2021). Krill-Oil-Dependent Increases in HS-Omega-3 Index, Plasma Choline and Antioxidant Capacity in Well-Conditioned Power Training Athletes. Nutrients, 13(12), 4237.
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2

Quantifying Oxidative Stress in Heart Tissue

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The formation of ROS in heart tissue lysates was measured by using 2,7-dichlorodihydrofluorescein diacetate (DCF-DA) (Invitrogen) (6 (link)) and Amplex Red as indicators according to the manufacturer’s instructions. The protein oxidation in heart tissues was assessed by measuring protein carbonyl content using a commercial assay kit (Cayman Chemical) according to the manufacturer’s instructions. The antioxidant capacity was measured based on reduction of copper (II) to copper (I) by using an OxiSelect Total Antioxidant Capacity Assay Kit (Cell Biolabs, Inc.).
Ni R., Zheng D., Xiong S., Hill D.J., Sun T., Gardiner R.B., Fan G.C., Lu Y., Abel E.D., Greer P.A, & Peng T. (2015). Mitochondrial Calpain-1 Disrupts ATP Synthase and Induces Superoxide Generation in Type 1 Diabetic Hearts: A Novel Mechanism Contributing to Diabetic Cardiomyopathy. Diabetes, 65(1), 255-268.
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3

Serum Total Antioxidant Capacity

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Serum TAC was determined applying the OxiSelect Total Antioxidant Capacity Assay Kit (Cell Biolabs, Inc.), according to the manufacturer's manual, by the ability of copper (I), as reduced from copper (II) by antioxidants, to react with a chromogenic reagent. Duplicate serum samples (20 μl), diluted 1:4 in PBS, were analyzed to provide concentrations in terms of copper-reducing equivalents, being proportional to the total reductive capacity.
Bousquet P.A., Meltzer S., Sønstevold L., Esbensen Y., Dueland S., Flatmark K., Sitter B., Bathen T.F., Seierstad T., Redalen K.R., Eide L, & Ree A.H. (2018). Markers of Mitochondrial Metabolism in Tumor Hypoxia, Systemic Inflammation, and Adverse Outcome of Rectal Cancer. Translational Oncology, 12(1), 76-83.
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4

Antioxidant Capacity Quantification Assay

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The TAC assay was performed using the OxiSelect™ Total Antioxidant Capacity Assay Kit as per manufacturer’s instructions (Cell Biolabs, Inc.). Briefly, increasing amounts of PIC1 or glutathione diluted in water were added to a 96 well microtiter plate. Titrating amounts of uric acid were also prepared in parallel to generate an antioxidant standard curve. Reaction buffer was then added and an initial absorbance recorded at 490 nm in a BioTEK plate reader. Next, copper ion reagent was added to each well, incubated for 5 minutes at room temperature followed by stop solution to terminate the reaction. The plate was then read again at 490 nm. To determine the total antioxidant capacity, mM uric acid equivalents extrapolated from the standard curve were converted to copper reducing equivalents as per the manufacturer’s instructions. Copper reducing equivalents are proportional to the sample’s total antioxidant capacity.
Gregory Rivera M., Hair P.S., Cunnion K.M, & Krishna N.K. (2018). Peptide Inhibitor of Complement C1 (PIC1) demonstrates antioxidant activity via single electron transport (SET) and hydrogen atom transfer (HAT). PLoS ONE, 13(3), e0193931.
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5

Rat Blood Biomarker Analysis

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On the past day of the experiment, all rats were anesthetized (as previously mentioned), and blood collected through cardiopuncture, and sera harvested by centrifugation at 3000 rpm for 10 min and stored at −20°C until analyzed. Blood samples were analyzed (platelet [PL] count, TC, triglycerides [TGs], and total antioxidant activity [TAO]- and LP) (ABX vet pack, 130312w3b, Horiba abx SAS, France; Cholesterol, 313AA; TG 230AA, Biosystem, Barcelona, Spain; Oxiselect total antioxidant capacity assay kit, 0628201304, Cell Biolabs Inc, USA; and TBARS assay kit, 1009055, Cayman Chemical, Michigan, USA), respectively.
Al-Tamimi H., Al-Dawood A., Awaishesh S, & Abdalla T. (2019). Resveratrol mitigates hypercholesterolemia exacerbated hyperthermia in chronically heat-stressed rats. Veterinary World, 12(2), 337-344.
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6

Antioxidant Capacity and ROS Quantification

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Total antioxidant capacity was determined by using the OxiSelect Total Antioxidant Capacity Assay Kit (Cell Biolabs, CliniSciences, Guidonia Montecelio, Italy), which relies on the principle that the reduction of divalent to monovalent Copper ion can form a stable-coloured complex with a chromogenic reagent. The net absorbance values measured at 490 nm in lysate samples derived from batches of 10 control and treated embryos at 30 hpf were compared with a uric acid standard curve and were proportional to the capacity of antioxidant species in samples. Each sample was assayed in duplicate, and results were expressed as μM Copper reducing equivalents.
ROS level was quantified by staining control and treated embryos in the dark for 15 min with 5 µM of the fluorescent dye 2,7-dichloro-dihydro-fluorescein-diacetate (DCFH-DA) green-fluorescent dye (Merck Life Science, Milan, Italy). After incubation, embryos were washed thrice with E3 medium and anesthetized as described above. The green fluorescence of oxidized DCF was visualized using a Leica M205-FA stereomicroscope and quantified using ImageJ software.
Reina C., Cardella C., Lo Pinto M., Pucci G., Acuto S., Maggio A, & Cavalieri V. (2023). Antioxidant, Pro-Survival and Pro-Regenerative Effects of Conditioned Medium from Wharton’s Jelly Mesenchymal Stem Cells on Developing Zebrafish Embryos. International Journal of Molecular Sciences, 24(17), 13191.
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7

Total Antioxidant Capacity Assay

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Total antioxidant capacity was measured using a purchased kit (OxiSelect™ Total Antioxidant Capacity Assay Kit, Cell Biolabs, Inc., San Diego, CA, USA), according to the manufacturer’s directions. Absorbance was read at 490 nm with a microplate reader (Synergy H1, Promega, Madison, WI, USA).
Chintala S.K., Pan J., Satapathy S., Condruti R., Hao Z., Liu P.W., O’Conner C.F., Barr J.T., Wilson M.R., Jeong S, & Fini M.E. (2023). Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy. International Journal of Molecular Sciences, 24(2), 981.
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8

Evaluating Liver Antioxidant Capacity

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The total antioxidant capacity of liver tissue was evaluated with a colorimetric OxiSelect Total Antioxidant Capacity Assay Kit (Cell Biolabs Inc., San Diego, CA, USA). Liver tissues were homogenized with the same volume of PBS. The homogenate was centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was collected and assays were performed according to the standard protocols.
Nakata R., Hyodo F., Murata M., Eto H., Nakaji T., Kawano T., Narahara S., Yasukawa K., Akahoshi T., Tomikawa M, & Hashizume M. (2017). In vivo redox metabolic imaging of mitochondria assesses disease progression in non-alcoholic steatohepatitis. Scientific Reports, 7, 17170.
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9

Serum TNFα and Urine Antioxidant Assessment

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Serum TNFα and urine total antioxidant capacity were measured using the Rat TNFα ELISA kit (Thermo Fisher Scientific, MA, USA) and Oxiselect Total Antioxidant Capacity Assay Kit (Cell Biolabs, CA, USA), respectively, according to the manufacturer’s protocol. For the TNFα measurement, 50 µL of serum was tested on the ELISA plate and its quantification was obtained by interpolating the difference of the absorptions at 450 nm and 550 nm in a standard curve, expressing the final result in pg/mL. The antioxidant capacity was measured by mixing the urine sample (20 µL) with 180 µL of the reaction buffer previously diluted in PBS 1X (1:100). The initial absorbance (490 nm) was read in order to calculate the net absorbance (final absorbance minus initial absorbance), and 50 µL of the copper ion reagent was added and then stirred for 5 min. The reaction was stopped by adding 50 μL of the stop solution and the final absorbance was read at 490 nm. The antioxidant capacity was calculated by comparing the net absorbance values obtained in duplicate with the standard curve. Values are shown in mM.
Jado J.C., Humanes B., González-Nicolás M.Á., Camaño S., Lara J.M., López B., Cercenado E., García-Bordas J., Tejedor A, & Lázaro A. (2020). Nephroprotective Effect of Cilastatin against Gentamicin-Induced Renal Injury In Vitro and In Vivo without Altering Its Bactericidal Efficiency. Antioxidants, 9(9), 821.
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10

Oxidative Stress Biomarkers in Plasma and Lung

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The levels of 3-nitrotyrosine (3NT); the concentration of reactive substances of thiobarbituric acid (TBARS), total antioxidant capacity (TAC) and nitrate/nitrite in plasma; and lung tissue homogenates were determined. Products of oxidative stress were evaluated using an OxiSelect™ Nitrotyrosine ELISA Kit (STA-305; Cell Biolabs Inc., San Diego, CA, USA) and an OxiSelect™ TBARS Assay Kit (STA-330; Cell Biolabs Inc., San Diego, CA, USA), and Cayman’s Nitrate/Nitrite Colorimetric Assay Kit (Alexis Corp., San Diego, CA, USA), and OxiSelect™ Total Antioxidant Capacity Assay Kit (STA-360; Cell Biolabs Inc., San Diego, CA, USA) kits were evaluated according to the manufacturer’s instructions.
Kosutova P., Nemcova N., Kolomaznik M., Mokra D., Calkovska A, & Mikolka P. (2022). Time-Dependent Oxidative Alterations in Plasma and Lung Tissue after Meconium Aspiration in a Rabbit Model. Antioxidants, 12(1), 37.
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