The method described by Erel (2004) was validated for use in cats. In this method, the blue–green colored oxidized 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is reduced to a colorless molecule, and the change in color is spectrophotometrically monitored. Reagent 1 was consisted of acetate buffer solution, pH 5.8, including 0.4 M CH3COONa (Sigma-Aldrich) and 0.36% glacial acetic acid (Sigma-Aldrich). Reagent 2 was consisted of acetate buffer, pH 3.6, containing 2 mM CH3COONa, 0.15% glacial acetic acid, 0.009% hydrogen peroxide (H2O2, Panreac Química SA) and 10 mM ABTS diammonium salt (Sigma-Aldrich). Two hundred fifty microliters of reagent 1 and 12 µl of serum sample were mixed and incubated at 37 °C for 150 s. Then, 25 µl of reagent 2 was added. Change in absorbance was monitored at 600 nm after 525 s. The method was adapted to an automated analyzer (Olympus 2700).
Abts diammonium salt
Manufactured by Merck Group
Sourced in United States, Italy
ABTS diammonium salt is a chemical compound used as a chromogenic substrate in various analytical and biochemical applications. It is commonly utilized in enzyme-linked immunosorbent assays (ELISA) and other colorimetric assays to measure enzyme activity or analyte concentration. The compound undergoes a color change when oxidized, enabling quantitative measurements.
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Lab products found in correlation
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (8 mentions)
2 2 azino bis, by Merck Group (6 mentions)
Gallic acid, by Merck Group (5 mentions)
Quercetin, by Merck Group (4 mentions)
Protocatechuic acid, by Merck Group (3 mentions)
Trolox, by Merck Group (3 mentions)
Sodium carbonate, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Potassium persulfate dipotassium peroxidisulfate 6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid trolox, by Merck Group (2 mentions)
Potassium persulphate, by Merck Group (2 mentions)
Luteolin, by Merck Group (2 mentions)
Folin ciocalteu s reagent, by Merck Group (2 mentions)
Hydroxytyrosol, by Merck Group (2 mentions)
Caffeic acid, by Merck Group (2 mentions)
Chlorogenic acid, by Merck Group (2 mentions)
Trifluoroacetic acid tfa, by Merck Group (2 mentions)
Bacto yeast extract, by BD (2 mentions)
Bacto tryptone, by BD (2 mentions)
L ascorbic acid, by Merck Group (2 mentions)
29 protocols using abts diammonium salt
1
Serum Antioxidant Capacity Assay in Cats
2
Antioxidant Evaluation of Natural Compounds
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Allopurinol, ascorbic acid, carminic acid (90% of purity), BHT, carmine (contains 42% of carminic acid), sodium carbonate, sodium hydroxide, acetic acid, citric acid, DPPH, potassium persulfate, ABTS diammonium salt, thiobarbituric acid, 1,1,3,3-tetraethoxypropane, hydrochloric acid, and trichloroacetic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).
3
ABTS Antioxidant Capacity Assay
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To evaluate total antioxidant capacity of 2-APB analogues, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay was conducted following a standard method provided by Sigma (EC 1.11.1.7) with minor modifications. ABTS assay is a common method used to detect total antioxidant capacity. While ABTS reacts with H2O2 catalyzed by peroxidase, ABTS●+ radical (an oxidized ABTS) is generated and a green color appears. Briefly, each well of the 96-well plate received 10 μL of KH2PO4 (100 mM, pH 5.0), 6 μL of H2O2 (1.66 mM), and various concentrations of 4 μL of 2-APB (or its analogues), mixed, incubated for 5 min at room temperature (RT), added with 40 μL of 9.1 mM ABTS diammonium salt (Sigma-Aldrich, Saint Louis, MO, USA) and 40 μL of 1 unit/mL peroxidase (Sigma-Aldrich, Saint Louis, MO, USA), and then incubated for additional 5 min at RT. The green color that formed through the reaction was observed, and the absorbance values were measured at 405 nm using a spectrophotometer (Multiskan EX; Thermo Scientific, Rockford, IL, USA). An ascorbic acid-treated group was used as a positive control. The % inhibition was calculated by the formula (A0 – A1)/A0 × 100%, where A0 is the absorbance of the control and A1 is the absorbance in the presence of a test or standard sample.
4
Antioxidant Compounds Quantification
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DPPH, ABTS diammonium salt, 2,4,6-tripyridyl-S-triazine (TPTZ), gallic acid, and quercetin were purchased from Sigma-Aldrich (MO, USA).
5
Biochemical Assays and Electron Microscopy
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The following chemicals were used in the experiment: bovine serum albumin (BSA), sucrose, tris hydroxymethyl aminomethane hydrochloride (Tris‐HCl), potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4), ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N’,N’‐tetraacetic acid (EGTA), N‐[2‐hydroxyethyl], piperazine‐N’‐[2‐ethanesulfonic acid], adenosine 5’‐diphosphate (ADP), proteinase K, CaCl2, succinic acid, EDTA, and a Bicinchoninic Acid Protein Assay Kit were purchased from Sigma Chemical Co. (St. Louis, MO); sodium succinate was purchased from Fisher Scientific (Fair Lawn, NJ). 4‐HNE and 4‐ONE were purchased from Cayman Chemical Co. (Ann Arbor, MI). Reagents for electron microscopy (glutaraldehyde and osmium tetroxide) were also purchased from Sigma Chemical Co. (St. Louis, MO). ABTS diammonium salt, horseradish peroxidase (≥250 units /mg), phosphate buffer solution (PBS), and ferrous sulfate heptahydrate (FeSO4.7H2O) were purchased from Sigma‐Aldrich (St. Louis, MO); H2O2 solution (30% w/w solution) and 2,4,6‐tri (2‐pyridyl)‐s‐triazine (TPTZ) were purchased from ACROS Organic (Morris, USA). Ferric chloride anhydrous (FeCl3) was purchased from Fisher Chemical (NJ, USA).
6
ABTS-Based Antioxidant Activity Assay
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The antioxidant activity was calculated by the azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) test30 (link). The ABTS diammonium salt, potassium persulfate (dipotassium peroxidisulfate), 6-hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and HPLC grade methanol were obtained from Sigma-Aldrich Milano, Italy. Trolox, 2.5 mM in methanol prepared day-to-day, was used as antioxidant stock standard. The ABTS and potassium persulfate were dissolved in distilled water to a final concentration of 7 and 2.45 mM, respectively, and mixed. The mixture was kept in the dark at room temperature for 16 h before its use to produce the ABTS radical (ABTS+). The ABTS radical solution was diluted using distilled water to have an absorbance of 1.00 at 734 nm. Samples (final concentrations 0.0001–0.0100 mg/ml) or Trolox standard (final concentration 0–20 mM) were added to the diluted ABTS+ solution; the absorbance was read 6 min after mixing by a UV/Vis spectrophotometer (Cary Varian, Palo Alto, CA, USA). Determinations were performed in triplicate and results expressed as the samples μmol Trolox equivalent antioxidant capacity (TEAC) ± SD.
7
Quantifying Peroxidase Activity in Cells
8
Serum Total Antioxidant Capacity Assays
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For determination of the serum TAS, ferric reducing antioxidant potential (FRAP) was performed as previously described [52 (link)], whereas the scavenging of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS●+) assay was employed as described by Gião et al. [53 (link)].
The ABTS●+ stock solution was prepared by reacting equal amount of 7 mM ABTS diammonium salt (Sigma-Aldrich, St. Louis, MO, USA) and 2.45 mM potassium persulphate (Merck, Damstadt, Germany). The reaction was developed for 16 h in the dark. Aliquots of serum samples (10 µL), diluted when needed, were added to 1 mL of ABTS●+ solution with an initial optical density (OD) of 0.70 ± 0.02 measured at 734 nm. After allowing the reaction to occur, the OD was recorded using an UV-Vis spectrophotometer (UVmini 1240, Shimadzu, Japan) and the results were calculated as inhibition percentage (IP) of ABTS●+, according to the equation ABTS●+ inhibition (%) = 100 − [(OD sample × DF)/OD ABTS] × 100, where OD sample indicates the sample absorbance following 6 min of reaction, DF is the dilution factor and OD ABTS refers to the initial absorbance of the diluted ABTS●+ solution. All measurements were performed in triplicate.
The ABTS●+ stock solution was prepared by reacting equal amount of 7 mM ABTS diammonium salt (Sigma-Aldrich, St. Louis, MO, USA) and 2.45 mM potassium persulphate (Merck, Damstadt, Germany). The reaction was developed for 16 h in the dark. Aliquots of serum samples (10 µL), diluted when needed, were added to 1 mL of ABTS●+ solution with an initial optical density (OD) of 0.70 ± 0.02 measured at 734 nm. After allowing the reaction to occur, the OD was recorded using an UV-Vis spectrophotometer (UVmini 1240, Shimadzu, Japan) and the results were calculated as inhibition percentage (IP) of ABTS●+, according to the equation ABTS●+ inhibition (%) = 100 − [(OD sample × DF)/OD ABTS] × 100, where OD sample indicates the sample absorbance following 6 min of reaction, DF is the dilution factor and OD ABTS refers to the initial absorbance of the diluted ABTS●+ solution. All measurements were performed in triplicate.
9
Determination of Antioxidant Compounds
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Folin–Ciocalteu’s reagent, potassium persulfate (K2S2O8) and standards of gallic acid were purchased from Merck (Darmstadt, Germany). Anhydrous sodium carbonate (Na2CO3) was acquired from Panreac (Castellar del Vallès, Barcelona, Spain). ABTS diammonium salt (2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), acetic acid and standards of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were obtained from Sigma-Aldrich (Madrid, Spain). Glycerol was acquired from Labbox (Barcelona, Spain), ultrapure water was obtained using a Milli-Q water system (Millipore, Milford, MA, USA) and ethanol was purchased from Romil Ltd. (Waterbeach, UK). High performance liquid chromatography (HPLC) grade acetonitrile was acquired from Honeywell Research Chemicals (Seelze, Germany), whereas the HPLC standards of hydroxytyrosol, protocatechuic acid, verbascoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, oleuropein and apigenin were purchased from Sigma-Aldrich (Madrid, Spain).
10
Antioxidant Activity Evaluation Protocol
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Q-3-G was obtained from Sigma Aldrich Co. (St. Louis, MO, USA). HaCaT, HEK293T, and B16F10 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). ABTS diammonium salt, DPPH, ascorbic acid (AA), ABTS, and retinol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), and phosphate-buffered saline (PBS) were bought from Gibco (Grand Island, NY, USA). 3-(4-5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Brisbane, Australia). TRIzol and PCR premix were purchased from Bio-D Inc. (Seoul, Korea). The cDNA synthesis kits were bought from Thermo Fisher Scientific (Waltham, MA, USA). Forward and reverse primers for PCR (polymerase chain reaction) were synthesized by Macrogen, Inc. (Seoul, Korea). All the antibodies related to the phosphorylated or total forms of the target protein were bought from Cell Signaling Technology (Beverly, MA, USA).
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