Infinite m2000 pro plate

The formation of Aβ fibrils was measured using Thioflavin T, an indicator of β-sheet formation, in a cell-free assay. Infinite M2000 Pro plate reader (Tecan). 10 μmol/L monomeric Aβ42 were incubated in a buffer consisting of Thioflavin-T (20 mmol/L in 150 mmol/L NaCl and 5 μmol/L of HEPES at pH 7.4), with and without 0.1 mg/mL HDL isolated by ultracentrifugation or an equivalent amount of apoB-depleted plasma based on HDL-C concentration, both from the same donor in a 96-well, black plate. The plate was kept at 37 °C and subjected to orbital shaking for 20 s every 5 min using an Infinite M2000 Pro plate reader. Fluorescence intensity was measured every 5 min for 12 h in total (440/490 nm excitation/emission).

The Trolox equivalent antioxidant capacity assay was used to compare the antioxidant activity of extracts towards the antioxidant effect of ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich). A radical cationic solution was used to measure the absorbance differences. Antioxidant extracts would inhibit the absorbance. ABTS^·+ (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate, Sigma Aldrich) was generated by oxidation while using potassium persulfate for at least 12 h. EtOH 96% (Merck KGaA, Darmstadt, Germany) was used to dilute the solution to an absorbance of 734 nm.
Trolox and extracts were diluted to concentrations that were between 0.1 mM and 0.00625 mM with 96% EtoH. For each run, solvent blank and positive controls with ABTS^·+ solution were performed. The absorbance was measured after 6 min. with an Infinite M2000 Pro™ plate reader (Tecan, Crailsheim, Germany). The decreased absorbance that was caused by Trolox was plotted for the different concentrations tested. The resulting plot was used as a calibration curve. The slope of the absorbance inhibition vs. antioxidant concentration plot was divided by the slope of the Trolox plot to calculate the Trolox equivalent antioxidant capacity (TEAC) as Trolox equivalents (TE) in mM/mg plant extract.