Spectrostar nano absorbance microplate reader

The antioxidant activity of the extracts was analyzed using the Ferric-reducing antioxidant power (FRAP) assay. In this assay, Fe³⁺ is reduced to Fe²⁺ by the antioxidant compounds and forms a colored complex with 2,4,6-tripyridyl-s-triazine in acetate buffer (pH 3.6) at 593 nm [23 (link)]. For the assay, the reactive solution was freshly prepared with 25 mL of 300 mM acetate buffer, pH 3.6, and 2.5 mL of 20 mM FeCl₃·6H₂O in deionized water, as well as 2.5 mL of 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl. The FRAP working solution (300 µL) was mixed with the acetonic algae extract (30 µL), and after 30 min, the absorbance was recorded by a SPECTROstar Nano absorbance microplate reader (BMG LABTECH, Ortenberg, Germany). The calibration was performed with astaxanthin (Sigma Aldrich, St. Lewis, MO, USA) in acetone, and the results are expressed in astaxanthin equivalent μmol_Ax·g⁻¹ lyophilized biomass.

The ferric reducing antioxidant power (FRAP) is based on the reduction of Fe³⁺ to Fe²⁺ by the antioxidant compound. A colored complex with 2,4,6-tripyridyl-s-triazine in acetate buffer with an absorption maximum at 593 nm is formed. The working solution was prepared with 25 mL of 0.3 M acetate buffer (pH 3.6), 2.5 mL of 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl, and 2.5 mL 20 mM FeCl₃·6H₂O in deionized water [50 (link),51 (link)]. The investigated compounds were dissolved in MeOH (100 mg mL⁻¹) and 50 µL were mixed with 300 µL FRAP working solution and subsequently the absorbance was recorded after 30 min (593 nm).
The second method is based on the scavenging of the ABTS radical 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). The radical cation was generated by reacting 3.5 mM ABTS with 1.2 mM K₂S₂O₈ in H₂O. Subsequently, the mixture was stored in darkness at room temperature for 12–16 h and then diluted with EtOH to a working solution (absorbance of 0.700 at 734 nm) before used. After the addition of ABTS working solution (300 µL) to the samples (50 μL), the decrease of the absorbance was recorded after 30 min [51 (link),52 (link),53 (link)].
For both assays the absorbance was recorded on a SPECTROstar Nano absorbance microplate reader (BMG LABTECH) and the calibration was carried out with ascorbic acid (Sigma Aldrich).

Plasma levels of LBP were determined by an LBP enzyme-linked immunosorbent assay kit for a wide variety of species (Hycult Biotech, Uden, Netherlands), according to the manufacturer’s instructions. Rat plasma samples were diluted 1:10 with dilution buffer prior to use in the analyses. The absorbance was read within 30 min at 450 nm in a SPECTROstar Nano Absorbance microplate reader (BMG Labtech, Ortenberg, Germany). Standard curves were generated using MARS data analysis software (BMG Labtech).