Fluostar optima plate reader

Primary cortical neurons were grown at a density of 8 × 10⁴ cells/cm² in poly-L-lysine treated plates for 9 days before treatment. PC12 cells were seeded at 4 × 10⁵ cells/well for 6 hours prior to treatment then treated with varying concentrations of H₂O₂ for 18 hours. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) was added at a concentration of 500 μg/mL to each well and incubated at 37°C for 4 hours. Media were discarded and cells were resuspended in 200 μL DMSO by repeated pipetting to solubilize the formazan. Suspensions were transferred to Eppendorf tubes and centrifuged at 12,000 g in a MiniSpin bench top microcentrifuge (Eppendorf) for 4 minutes. 50 μL supernatant was transferred in triplicate to the wells of 96-well plate and absorbance was measured at 590 nm on a Fluostar Optima plate reader (BMG Lab Technologies).

The ACE-inhibitory activity of synthetic peptides was determined by the fluorescence protocol optimized by Sentandreu and Toldrá (2006) [18 (link)] and modified by Quirós et al. (2009) [19 (link)]. Briefly, the substrate Abz–Gly–Phe(NO₂)–Pro was dissolved (0.45 mM) in 150 mM Tris and 1125 mM NaCl buffer (pH 8.3) and maintained at 4 °C until its use. ACE (1 U/mL) was diluted (0.04 U/mL) in 150 mM Tris buffer containing 0.1 μM ZnCl₂ (pH 8.3). Forty microliters of sample (or MilliQ water for blank and control) were added to a black multi-well plate (Porvair, Leatherhead, UK). Then, 40 μL of ACE were added, and the reaction started after the addition of 160 μL of the substrate. The plate was incubated at 37 °C for 30 min, and the fluorescence was measured in a FLUOstar OPTIMA plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 320 nm excitation and 420 nm emission filters. Data were processed with the FLUOstar Control version 1.32 R2 (BMG Labtech) software and expressed as IC₅₀ (peptide concentration needed to inhibit 50% of the ACE activity).

As a proxy for antioxidant capacity, we assayed oxygen radical absorbance capacity (ORAC) according to [37 (link)] with minor modifications; the measured values were normalized to the individual protein content. This biomarker represents the water-soluble fraction of antioxidants and has been applied for analysis of antioxidant production in daphnids [38 (link)]. Samples for ORAC and protein measurements were homogenized in 100 μL of PPB buffer (75 mM, pH 7.4). Fluorescein was applied as a fluorescent probe (106 nM) and 2, 2- azobis (2-amidinopropane) dihydrochloride (AAPH) (152.66 mM) as a source of peroxyl radicals. Trolox (218 μM, Sigma–Aldrich) was used as the standard. The assay was conducted in 96-well microplates while 20 μL of homogenate sample was added to each well and mixed with 30 μL of AAPH and 150 μL of fluorescein. Fluorescence was measured at 485nm/520nm (excitation/emission wavelength).
Protein content of the supernatant was determined by the bicinchoninic acid method using a Pierce BCA Protein Assay kit 23227 (ThermoFisher, USA) according to the microplate procedure with some modifications. In each well, 25 μl of blank, standard or samples was added to 200 μl of working solution. Absorbance was measured at 540 nm using a FluoStar Optima plate reader (BMG Lab Technologies, Germany). Antioxidant capacity was expressed as mg Trolox eq. mg protein⁻¹.

Thioflavin T (ThT) binds to β sheet rich structures present in amyloid fibrils, with fluorescence increasing proportionally to the quantity of fibrils present in solution. ThT (final concentration 100 μM) was added to wells in a Greiner 96-well plate together with αSA53T (100 μM), in the absence or presence of each test compound (either at 200 or 100 μM) in 50 mM ammonium acetate buffer to a total volume of 100 μl in each well. Fluorescence was measured at 37°C every 30 min for 100 h using a Fluostar Optima plate reader (BMG Lab technologies, Australia) with a 440/490 nm excitation/emission filter. The ThT assay was performed in duplicate and repeated three times. Results were normalized to blank values (ThT alone in 50 mM ammonium acetate buffer).