Genios plus

The Oxygen Radical Absorbance Capacity (ORAC) of PINO was assayed as described in Prior et al. [30 (link)]. This method measures the oxidative degradation of fluorescein induced by the thermal decomposition of the AAPH azo-compound. In brief, fluorescein (48 nM) was added to each well of a round bottom 96-well plate that was previously tempered at 37°C. Then, PINO (from 0.001 to 1000 μM), Trolox^TM (standard, from 12.5 to 100 μM) or PBS (blank) with a final volume of 1 % DMSO (v/v) was added to the wells. After incubating for 15 min at 37 °C, AAPH was added to the wells. Fluorescence readings (Ex. λ₄₈₅/Em. λ₅₂₀ nm) were taken every 5 min at 37 °C for 160 min with a TECAN GENios Plus microplate reader. The final results were calculated based on the difference in the area under the fluorescence decay curve (AUC) between the blank and each sample. The AUC formula was determined as follows: $AUC=1+f_1/f_0+f_2/f_0+f_3/f_0+\dots +f_n/f_0$
where f₀ is the initial fluorescence at cycle 0 and f_n is the fluorescence reading at cycle n.
The results were expressed as micromolar Trolox^TM equivalents (TE), which were calculated using the line equation from the standard curve: $TE=\left(Y–b\right)/m$
where Y is the net AUC (AUC_sample – AUC_control), b is the Y-intercept and m is the slope.

For the quantitation of caspase-3/7 activity, a luminescent Caspase-Glo-3/7 Assay (Promega) was used according to the manufacturer’s instructions and previously described method [13 (link)]. Luminescence is proportional to the amount of caspase activity present and, for the purpose of comparison, 10 µg total protein was utilized. Luminescence was measured using a microplate reader GENios Plus (TECAN). Z-DEVD-FMK (100 μM), a specific caspase-3 inhibitor, was also used.

To estimate the effects on cell viability, CellTiter-Blue^® Cell Viability Assay from Promega (G8081, Mannheim, Germany) was performed. In 96-well plates 5 × 10³ cells of the different cell lines (except 7.5 × 10³ cells of HepG2 and Capan-2 due to slower growth rates) were seeded in a volume of 100 µL as pentaplicates, incubated overnight, and after removing the medium, exposed to medium containing 0.1, 1, 10, or 100 µM of cis- or trans-resveratrol, or 10 µM SAHA as positive control. After 72 h incubation, 20 µL of assay reagent were added and gently mixed. Measurement of fluorescence was carried out after empirically determined incubation times at 37 °C for each cell line (1.5 h for MiaPaCa; 1.75 h for A498; 2 h for HepG2, Capan-2, HCT-116, HCT-116/p53^(−/−), SN12C; 4 h for Hep3B) with a microtiter plate reader (GENios Plus, Tecan, Crailsheim, Germany; recorded fluorescence (550_Ex/595_Em), adjusted gain: 60). CTB assay was repeated in three independent experiments, respectively.

Cells were seeded in 24-well plates and infected 48 h later. At 96 hpi, cells were washed with ice-cold PBS, fixed with 10% trichloroacetic acid (TCA), and incubated at 4 °C for 30 min. Then, TCA was removed, and the fixed cells were washed with tap water and dried for 24 h. To stain cells, 250 µL of SRB dye (0.4% dissolved in 1% acetic acid) was added followed by incubation at RT for 10 min, and cells were washed with 1% acetic acid and dried again. Protein-bound dye was dissolved in 10 mM Tris base for 10 min before measurement of optical density was performed in a 96-well microtiter plate reader (Tecan Genios Plus, Tecan Deutschland, Crailsheim, Germany) at a wavelength of 550 nm (reference wavelength of 620 nm). The SRB assay allows densitometric quantification of the total cellular protein mass after incubation of the cell mass of interest with cytotoxic substances for a distinct time range; thereby, a remnant cell mass is calculated which represents the cytotoxic effectiveness measured under the respective experimental conditions.

Laccase activity was routinely determined following the oxidation of 2 mM ABTS in McIlvaine buffer (pH 4.0) at 420 nm, as previously described (Junghanns et al., 2008b (link); Hofmann and Schlosser, 2016 (link)). Peroxidase activity was determined following the oxidation of 2 mM ABTS in presence of 100 μM H₂O₂ and 1 mM ethylenediaminetetraacetate (EDTA) disodium salt in 50 mM sodium malonate buffer (pH 4.5) (peroxidase procedure modified from Schlosser et al., 2000 ). Peroxidase activities were corrected for laccase activities through omitting H₂O₂. Enzyme activities are expressed in international units (U), where 1 U is defined as the amount of enzyme capable of oxidizing 1 μmol ABTS per minute. All enzymatic assays were performed using a GENios Plus microplate reader (Tecan, Männedorf, Switzerland).

Cell metabolic activity was evaluated using the oxidizable and reducible colorimetric probes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [23 (link)]. Cells were treated as indicated in figure legends and then processed for the MTT assay. After treatments, cells were added with 20 µL MTT solution (5 mg/mL) in medium M199 and placed at 37 °C in a cell incubator for 4 h. After that, the medium was discarded, and the converted dye was solubilized with acidic isopropanol (0.04 N HCl in absolute isopropanol), and the multi wells were read at 570 nm using a GENios plus microplate reader (Tecan) with background subtraction at 650 nm. Results were expressed as a percent of untreated control cells. Cell viability was calculated by the following equation: Cell viability (%) = (Abs of sample/Abs of control) × 100, where Abs of sample is the absorbance of the cells incubated with the different concentrations of the two extracts, and Abs of control is the absorbance of the cells incubated with the culture medium only (positive control).

For cytotoxicity assessment on cross-linked matrices, samples eluates were obtained, according to UNI EN ISO 10993-5, by incubating the samples (i.e., EDC/NHS, and GTA solution cross-linked matrices) in culture medium for 1 h and 24 h. The medium was composed by Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) 10% v/v, penicillin/streptomycin 1% v/v, glutamine 2 mM, Hepes 10 mM and sodium pyruvate 1 mM.
HeLa cells (epithelial cell line from cervix carcinoma) were seeded at a density of 10⁴ cells/well in 96-wells plates and cultured with fresh complete medium until 70% confluent. The medium was then replaced with eluates or control (i.e., medium aged for the same time) and cells were returned to the incubator. After 24 h, cell metabolic activity was assessed by Alamar Blue™ assay, according to manufacturer recommendations, and the fluorescence of each sample was spectrophotometrically read in a multiwell plate reader (Tecan, Genios Plus, Mannedorf, Switzerland).