The antioxidant activity was evaluated by Trolox Equivalent Antioxidant Capacity (TEAC) method adapted for 96-well microplates and Infinite M200 (Tecan, Männedorf, Switzerland), using the radical cation ABTS•+ and Trolox (Hoffman-La Roche) as standard [104 (link),105 (link)]. Briefly: 10 µL of each sample was added to 200 µL of ABTS•+ solution, were stirred and the absorbance at 734 nm was read at 6 min [11 (link),106 (link)]. Trolox was used as standard and was assayed under the same conditions of the samples. Results were expressed as nmol of Trolox Equivalents per mg of contained proteins (nmol TE/mg protein).
Trolox
Manufactured by Roche
Sourced in Switzerland
Trolox is a water-soluble vitamin E analog used as an antioxidant in biochemical and cell culture applications. It functions as a free radical scavenger to prevent oxidative damage.
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Lab products found in correlation
Infinite m200, by Tecan (1 mentions)
Sodium ascorbate, by Merck Group (1 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Sodium azide, by Roche (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Black 96 well plate, by Thermo Fisher Scientific (1 mentions)
Gen5 software version 1, by Agilent Technologies (1 mentions)
Infinite 200, by Tecan (1 mentions)
5 protocols using trolox
1
Trolox Equivalent Antioxidant Capacity Assay
2
Proximity Labeling of Cyclin D1 EV Targets
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Cylin D1–APEX N2A cells were used to capture the proximity labeling reaction or collect purified cylin D1–APEX EVs. For the APEX reaction in receipt mESCs, cylin D1–APEX EVs were incubated with mESCs in N2B27 medium for 2 d at a concentration of ∼5 × 109 EVs/ml medium. APEX proximity labeling was conducted as described previously (Hung et al., 2016 (link)). Biotin-phenol (500 µM) was preincubated with cells for 30 min at 37°C. Immediately before use, 1 mM (0.003%) H2O2 (Thermo Fisher Scientific) was spiked into the medium for the 1-min labeling reaction at RT. The reaction was then quenched immediately by three thorough washes with RT quencher solution, containing 10 mM sodium ascorbate (Sigma-Aldrich), 5 mM Trolox (Sigma-Aldrich), and 10 mM sodium azide (Sigma-Aldrich) in Dulbecco’s PBS (Thermo Fisher Scientific). Cells were lysed in radioimmunoprecipitation assay (RIPA) medium (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS, pH 7.4) supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, 1 mM Trolox, 1 mM DTT, and protease inhibitors (Roche). The whole-cell lysate was combined with loading buffer, heated at 95°C for 10 min, and resolved by SDS-PAGE. Biotinylated proteins were evaluated by blotting with streptavidin-HRP (21130; Thermo Fisher Scientific).
3
Colorimetric Determination of Total Antioxidant Status
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The total antioxidant status (TAS) of the blood serum of patients and healthy subjects was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) [16 (link)]. For this purpose, a solution ABTS cationic radical was prepared in phosphate buffer (PBS, pH 7.4). 3 times diluted blood serum samples (5 μL) were incubated (37 °C) with ABTS radical working solution (245 μL) for 10 min. Absorbance was measured using an Infinite 200 plate reader (TEKAN, Switzerland) at 734 nm.
The test results are reported with reference to the equivalent antioxidant capacity of Trolox (TEAC) as a standard. Trolox (Hoffman-LaRoche, Basel, Switzerland) is a water-soluble derivative of vitamin E with antioxidant properties and does not interact with other cellular components. The TAS level is therefore expressed in mmol Trolox/l.
The test results are reported with reference to the equivalent antioxidant capacity of Trolox (TEAC) as a standard. Trolox (Hoffman-LaRoche, Basel, Switzerland) is a water-soluble derivative of vitamin E with antioxidant properties and does not interact with other cellular components. The TAS level is therefore expressed in mmol Trolox/l.
4
Oxygen Radical Absorbance Capacity Assay
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The AA was determined by the oxygen radical absorbance capacity (ORAC) method described previously [15 (link)]. Briefly, the reaction was performed at 37 °C in 75 mM phosphate buffer at pH 7.4. The reaction mixture (200 μL) contained 180 μL of 70 nM fluorescein, 90 μL of 12 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 30 μL of diluted sample or standard (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox, Hoffman-LaRoche, basel, Switzerland) at concentrations ranging from 1 to 160 μM. Reaction mixtures were placed in a black 96-well plate (Fisher Scientific, Hampton, VA, USA) and the fluorescence was read in a Synergy HT microplate reader (BioTek Instruments, Winowski, VT, USA) every minute at excitation and emission wavelengths of 485 and 520 nm, respectively. The equipment was controlled by Gen5™ software, version 1.1 (BioTek Instruments). Results were expressed as μmol Trolox equivalents (TE)/g dw.
5
Antioxidant Capacity of Keratinocytes
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Total antioxidant status (TAS) of keratinocytes was determined with a commercial kit using the 2,2-azinobis-(3-ethybenzodiazoline) 6-sulfonic acid (ABTS) radical cation assay according to the manufacturer’s protocol (Randox, Kearneysville, WV, USA). The ABTS test allows for determining the total effectiveness of lipophilic and hydrophilic antioxidants [84 (link)]. Pre-generated with oxidation of ABTS (2,29-azinobis-(3-ethyl-benzothiazoline- 6-sulfonic acid) by potassium persulfate, the colored radical monocation 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+•) was reduced in the presence of hydrogen-donating antioxidants. The reduction of ABTS+• reflects the antioxidant potential of the sample. For this purpose, the keratinocyte lysate was added to the ABTS+• working solution in a 96-well plate at 37 °C and the absorbance reduction was analyzed at 734 nm (Infinite 200; TECAN, Männedorf, Switzerland) for 10 min. The test results are reported with reference to the equivalent antioxidant capacity of Trolox (TEAC) as a standard. Trolox (Hoffman-LaRoche, Basel, Switzerland) is a water-soluble derivative of vitamin E with antioxidant properties and does not interact with other cellular components. The TAS level is therefore expressed in µmol of Trolox per mg of protein.
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