Oxygen Radical Absorbance Capacity Assay

The oxygen radical absorbance capacity (ORAC) assay followed the protocol outlined by Coscueta et al. (2019) [52 (link)]. A black polystyrene 96-well microplate (Nunc, Roskilde, Denmark) was utilized, and measurements were taken using a Multidetection plate reader (Synergy H1, Vermont, USA) controlled by Gen5 Biotek software version 3.04. Fluorescence was monitored for 80 min at 1 min intervals. Each sample, standard, blank, or control analysis was duplicated. The final ORAC values were expressed as Trolox Equivalents (μmol TE).

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Fernandez Cunha M., Coscueta E.R., Brassesco M.E., Marques R., Neto J., Almada F., Gonçalves D, & Pintado M. (2023). Exploring Bioactivities and Peptide Content of Body Mucus from the Lusitanian Toadfish Halobatrachus didactylus. Molecules, 28(18), 6458.

Publication 2023

black polystyrene 96-well microplate Synergy H1

Assay Fluorescence Oxygen radical absorbance capacity Polystyrene Trolox

Corresponding Organization :

Other organizations : Universidade Católica Portuguesa, ISPA - Instituto Universitário, University of Saint Joseph

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Variable analysis

independent variables

Oxygen radical absorbance capacity (ORAC) assay

dependent variables

Fluorescence monitored for 80 min at 1 min intervals

control variables

Black polystyrene 96-well microplate (Nunc, Roskilde, Denmark)
Multidetection plate reader (Synergy H1, Vermont, USA) controlled by Gen5 Biotek software version 3.04
Each sample, standard, blank, or control analysis was duplicated

controls

Blank
Control

Annotations

Based on most similar protocols

Use a black polystyrene 96-well microplate to minimize background fluorescence (Input protocol)

Monitor fluorescence for 80 minutes at 1 minute intervals (Input protocol)

Duplicate each sample, standard, blank, or control analysis for improved reproducibility (Input protocol)

Use Trolox (7.5-240 μM) as a standard for calibration, with a correlation coefficient of 0.99 (Protocol 1)

Incubate the reaction mixture (fluorescein, Trolox/sample, AAPH) at 37°C (Protocols 2, 4)

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