Black round bottom 96 well microplates

The procedure was modified from the method described by Ou et al. [17 (link)], using Trolox as a control standard. The ORAC assay was carried out in black round bottom 96-well microplates (Costar) and absorbance was measured with an automated plate reader (Fluostar Optima, BMG Labtech). All the samples (extracts, fluorescein, and AAPH) were diluted in 75 mM phosphate buffer (pH 7.4). Thirty microliters of each extract (1 mg/mL) or phosphate buffer (blank) was mixed with 180 μL of fluorescein solution (117 nM final concentrations) and incubated for 5 min at 37°C. A volume of 90 µL of AAPH solution (40 mM final concentration) was added and fluorescence was immediately monitored using 485 nm excitation and 520 nm emission wavelengths at 1 min intervals for 70 min. The antioxidant capacities of the extracts were expressed as mg of Trolox equivalent per g of extract (mg of TE/g of E). All samples were analyzed in quadruplicate and at least in three different experiments.

The procedure was modified from the method described by Ou et al. (2001[29 (link)]). The ORAC assay was accomplished in black round bottom 96-well microplates (Costar) on a Fluoroskan Ascent FL™ plate reader (Labsystems) using an automated injector. The test was directed at 37.5 °C and in pH 7.4 phosphate buffer, with a blank sample in parallel. Different concentrations of Trolox as the control standard were used in quadruplicate, and a gradient of 16 concentrations of the extracts was set without replication. The fluorimeter was planned to register the fluorescence (λ ex.: 485 nm/em.: 530 nm) of fluorescein each minute once adding of 375 mM of 2,2-azobis (2-amidinopropane) dihydrochloride (AA-PH), for 35 min. The final results were calculated using the net area under the curves of the extract concentrations for which reduction of at least 95 % of fluorescence was detected at 35 min and which also showed a linear dose-response pattern. ORAC values were stated in micromoles of Trolox equivalents (TE) per gram (µmol TE/g).

The procedure was modified from the method described by Ou et al. (2001 (link)), using Trolox as a control standard. The ORAC assay was carried out in black round-bottom 96-well microplates (Costar) and absorbance was measured with an automated plate reader (Fluostar Optima; BMG Labtech). All the samples (extracts, pure polyphenols, fluorescein and AAPH) were diluted in 75 mM phosphate buffer (pH 7.4). Thirty microliters of extracts (1 mg/mL) or phosphate buffer (blank) were mixed with 180 μL of fluorescein solution (117 nM final concentration) and incubated for 5 min at 37 °C. A volume of 90 μL of AAPH solution (40 mM final concentration) were added and fluorescence was immediately monitored using 485 nm excitation and 520 nm emission wavelengths at 1 min intervals for 70 min. The antioxidant capacities of extracts or purified molecules were expressed as mg of Trolox equivalent per g of extract (mg TE/g) or mmol equivalent Trolox per g of polyphenols (mmol TE/g P), respectively. All samples were analyzed in quadruplicate and at least in three different experiments.