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Total antioxidant capacity assay kit

Manufactured by Abcam
Sourced in United Kingdom, United States

The Total Antioxidant Capacity Assay Kit is a colorimetric assay designed to measure the total antioxidant capacity in a variety of biological samples. The kit utilizes a copper(II) reduction reaction to quantify the total antioxidants present in the sample.

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27 protocols using total antioxidant capacity assay kit

1

Antioxidant Capacity in Pah-R261Q Liver

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The antioxidant capacity of small molecules (such as ascorbate, glutathione, and vitamin E) in Pah-R261Q and WT liver homogenates was determined by the Trolox equivalent antioxidant capacity assay using the colorimetric Total Antioxidant Capacity Assay kit (Abcam). Samples were diluted 1:1 with the included reagent Protein Mask to avoid the potential contribution of other biological species. Otherwise, the standard protocol indicated in the product manual was followed. Results were obtained by interpolation to a Trolox (reference antioxidant) standard curve and expressed as Trolox equivalent antioxidant capacity.
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2

Antioxidant Capacity of Omega-3 in Alzheimer's

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To measure total antioxidant capacity of H19-7 cells affected by ω-3 fatty acids with Aβ1-40 induction, cells were pretreated with different ω-3 fatty acids (100 μM) for 48 h followed by 24 h induction with Aβ1-40. After washing with PBS, the total antioxidant potential of samples was determined spectrophotometrically at 570 nm by using a Total antioxidant capacity assay kit (Abcam, Cambridge, UK) according to manufacturer’s instructions. These kit measures combined nonenzymatic antioxidant capacity. Briefly, both small molecules and proteins that carry anti-oxidant capacity are able to convert Cu2+ ion to Cu+ ion. The reduced Cu+ ion is chelated with a colorimetric probe that will display a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity. A standard concentration of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used to create a calibration curve and the results of the assay were expressed as nanomoles per microliter Trolox equivalents. Values were normalized to the protein content.
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3

Quantification of Oxidative Stress Markers

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The quantification of 8-OHdG, 8-isoprostane, and TAC in urine samples was performed in accordance with the respective manufacturer’s instructions (8-OHdG ELISA kit, Biovision, Waltham, MA, USA; 8-isoprostane ELIZA kit, Enzo, Farmingdale, NY, USA; Total Antioxidant Capacity Assay Kit, abcam, Cambridge, MA, USA). The laboratory procedures were similar to those reported in a previous study [19 (link)].
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4

Measuring RBC Antioxidant Capacity

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RBC pellet was transferred to a clean tube with 1 * 107 RBC/ml in 0.1 mol PBS (pH 7.4), snap frozen and stored at −80 °C until measurement. Samples were thawed on ice and the total antioxidant capacity assay kit (Abcam, UK) was applied to measure RBC total antioxidant capacity according to the manufacturers´ instructions.
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5

Oxidative Stress Biomarkers Evaluation

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All assays were analyzed on a Tecan Sunrise microplate reader. The markers of inflammation/oxidative stress that we measured were as follows: 8-hydroxy-2’-deoxyguanosine (8-OHdG) (Trevigen, Gaithersburg, MD), protein carbonylation concentration (OxiSelect Protein Carbonyl ELISA Kit, Cell BioLabs), small molecule antioxidant capacity (Total Antioxidant Capacity Assay Kit, Abcam), catalase activity (StressXpress Colorimetric Activity Kit, StressMarq Biosciences Inc), and ferritin (Ramco Laboratories, Stafford, TX) according to the manufacturers’ protocol.
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6

Antioxidant Capacity Assay in Endothelial Cells

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The total anti-oxidant capacity was measured in HBMECs and OECs using a total antioxidant capacity assay kit (Abcam, Cambridge, UK) as per manufacturer’s instructions. In brief, cells grown under normal and experimental conditions were pelleted by trypsinisation which were then re-suspended with double distilled water (ddH2O) before centrifugation for 4 min at 10,000 rpm to obtain the supernatant. 100 µL of working solution (the mixture of Cu2+ reagent and assay diluent) was then mixed with samples and standards and incubated at room temperature for 90 min before measuring total anti-oxidant capacity in a plate reader (FLUOstar Omega, BMG Labtech, Aylesbury, UK) at 570 nm.
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7

Measuring Systemic Antioxidant Capacity

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To assess the systemic antioxidant capacity, blood TAC was measured using a commercially available kit (Total Antioxidant Capacity Assay Kit, Abcam plc, Cambridge, UK). This colorimetric method is based on the reduction of Cu2+ to Cu+ by smaller oxidative molecules and proteins. Because the reduced Cu+ ion is chelated with a colorimetric probe with an absorbance peak around an optical density of 570 nm, an optical density at 570 nm is proportional to the TAC. All standards and samples were measured in duplicate. A reference curve was constructed using the antioxidant capacity of known concentrations of trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and the antioxidant capacity of the examined sample was expressed in trolox equivalents antioxidant capacity (TEAC).
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8

Antioxidant Capacity Evaluation in Cells

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To evaluate the antioxidant effect in both medium and neutrophils, we utilized the Total Antioxidant Capacity Assay Kit from Abcam (Cat. No. ab65329, Cambridge, UK) following the manufacturer’s protocol without the protein mask. The TAC was assessed in the culture media components: RPMI 1640 alone, AS alone, and RPMI 1640 + AS, following the manufacturer’s instructions for liquid samples. For the evaluation of TAC in neutrophils, we seeded 1 × 106 neutrophils and followed the same protocol described in the section of NETosis assay for the controls and the combination of antioxidants GSH + NAC and ALL. After inducing NETosis, in all cases, the cell suspensions were washed twice with cold PBS 1× to remove the media. Subsequently, the samples were resuspended in 100 µL of ddH2O containing 0.05% Triton X-100 (Cat. No. 85111, Thermo Fisher). The resulting homogenate was then incubated on ice for 10 min. After incubation, the samples were centrifuged at top speed for 5 min at 4 °C. The supernatant was collected, and the assessment was carried out according to the instructions provided in the kit. The output of the TAC assay was measured using a microplate reader (ALLSHENG, Hangzhou, China) at 570 nm.
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9

Total Antioxidant Capacity Measurement

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Reduced Cu2+ was measured as marker for the total antioxidant capacity (Total Antioxidant Capacity Assay Kit [Abcam]) in the cell culture medium of HUVEC (diluted 1:4 with medium), in lysed HUVEC, in plasma (diluted 1:100 with PBS), and in lysed RBC (1:10,000). Cell lysis was achieved via ultrasound and vortex. Both, Trolox standard (0, 4, 8, 12, 16, 20 nmol) and samples were incubated with a Cu2+ reagent for 90 min at RT on an orbital shaker and the absorbance was read at 570 nm using the Multiskan FC Microplate Photometer (Thermo Fisher Scientific). The absorbance was plot as a function of Trolox concentration, and the sample antioxidant Trolox equivalent concentrations were calculated by linear regression.
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10

Antioxidant Capacity Measurement

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This was measured using the total antioxidant capacity assay kit provided by Abcam according to the method described by Suresh et al. [14 (link)].
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