Victor x2

Neutralization assays were carried out using TZM-bl cells as described before [37 (link)]. Briefly, Env-pseudotyped viruses were pre-incubated in 96-well tissue culture plates with various concentrations of bnAbs (IgG) for an hour at 37° C in a CO₂ incubator under humidified conditions. Subsequently, 1 × 10⁴ TZM-bl cells were added to the mixture in the presence of 25 µg/ml DEAE-dextran (Sigma, Inc.). The plates were further incubated for 48 h. The degree of virus neutralization was assessed by measuring reduction in relative luminescence units (RLU) in a luminometer (Victor X2; PerkinElmer Inc.).

After 24 h of transfection, A2780 and SKOV3 cells were seeded in 96 well culture plates (2 × 10³ cells/well). Then, 20 μM cisplatin and a control medium were added for an additional 24 h. For dose-dependent experiments, the cells were separately cultured with cisplatin at 0, 10, and 20 μM for 24 h. CCK-8 test solution (Bimake, Houston, TX, USA) was used to detect cell proliferation in response to cisplatin. After one hour of incubating, cell viability was determined by recording the optical density values at a test wavelength of 450 nm utilizing a VICTOR X2 microplate reader (PerkinElmer, Waltham, MA, USA).

The ability of compounds to competitively displace propidium iodide was evaluated by a fluorescence method⁴³ . To determine the degree of displacement (% displacement) of propidium iodide from the PAS of AChE, EeAChE (fnal concentration 7 µM) was incubated with the test compound at a concentration of 290 and 340 µM in 1 mM Tris-HCl bufer pH 8.0, 25 °C for 15 min. Then, propidium iodide solution (final concentration 8 µM) was added, the samples were incubated for 15 min and the fluorescence spectrum 530 nm (excitation) and 600 nm (emission) was taken. Donepezil was used as reference compound. The blank contained propidium iodide of the same concentration in 1 mM Tris-HCl buffer pH 8.0. The measurements were carried out in triplicate on a microplate reader Perkin Elmer VictorX2 (Perkin Elmer, Singapur).

Neutralization assays were carried out using TZM-bl reporter cells as described before [69 (link)]. Briefly, Env-pseudotyped viruses were incubated with varying dilutions of antibodies (mAbs, serum and plasma) for 1 h at 37°C in a CO₂ incubator under humidified condition. TZM-bl cells (1 X 10⁴) were added into the mixture virus-antibody mixture containing 25 μg/ml DEAE-dextran (Sigma). The plates were further incubated for 48 h and the extent of virus neutralization was assessed by measuring relative luminescence units in a luminometer (Victor X2, PerkinElmer Life Sciences).

Target Calu-3 cells were seeded in culture medium the day before infection (7 × 10⁴) in 24 wells plates. Complete culture medium (DMEM-F12 supplemented with 10% FBS) was removed, and cells were inoculated with SARS-CoV-2 pseudovirus (in DMEM-F12 no FBS) at a multiplicity of infection (MOI) of 0.05 for 1 h at 37 °C in a humidified incubator. One hour post infection, the medium containing the SARS-CoV-2 pseudovirus was removed, cells were washed in 1x PBS, and fresh compete medium was added to each well. Infection efficiency was quantified 16 hpi by measuring the activity of firefly luciferase in cell lysates using a commercial substrate (Cod. #6066766; Britelite plus, PerkinElmer Italia, Milan, Italy) in a plate luminometer (VictorX2, PerkinElmer, Milan, Italy). Luciferase values were normalized by the cellular protein content (Cod. #23227; Pierce™ BCA Protein Assay Kit, ThermoFisher Scientific, Rodano, Milan, Italy).

The neutralizing ability of the fraction against the oxygen-derived radicals measured by ORAC Assay [87 (link)] was performed employing plate reader Perkin Elmer Victor X2 (Cridersville, OH, USA). Dilution of the bioactive and the analysis was performed in phosphate buffer pH 7.4. For the reaction: 45 µL of the diluted sample (1:40) was mixed with 175 µL fluorescein (108 nM in well), then 50 µL (18 mM in well) of freshly prepared AAPH as a source or the peroxyl radical were added to 96-well plates with black flat bottom wells. Fluorescence conditions were as follows: 37 °C, excitation at 485 nm and emission at 535 nm, for 5 h.