Xmark microplate spectrophotometer

Plasma creatinine level was measured by using a LabAssay^TM Creatinine assay kit (Fujifilm Wako Chemicals) according to the manufacturer's instructions. Briefly, plasma samples were deproteinized (sodium Tungstate and phosphoric acid) and centrifuged at 600 g for 10 min at room temperature. One hundred microliters of supernatant was then mixed with 50 µl of picric acid (22 mM) and 50 µl of NaOH (0.75 M) and incubated at 25–30°C for 20 min. The absorbance at 520 nm was then read within 30 min by a Bio‐Rad xMark microplate spectrophotometer (Bio‐Rad) and plotted against a standard curve. Data are expressed as mg/dl.

The in vitro cytotoxicity was determined by means of (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. In brief, HaCaT, 1BR3 cells were seeded in 96-well plates at an initial density of 1104 cells/well and allowed to attach. Next, the old medium was removed and a fresh one was added containing five concentrations (100 µM, 10 µM, 1 µM, 0.1 µM, and 0.01 µM) of the tested compounds 6 and 7 after which cells were incubated for 48 h. The control cells were treated with the same amount of DMSO, the highest concentration of DMSO present in the medium being 0.5%. A volume of 10 mL MTT reagent (5 mg/mL) was added in each well. During a 4 h contact period, the intact mitochondrial reductase converted and precipitated MTT as blue crystals. The precipitated crystals were dissolved in 100 mL of lysis solution provided by the manufacturer (Sigma-Aldrich). Finally, the reduced MTT was spectrophotometrically analyzed at 570 nm, using a microplate reader (xMark Microplate Spectrophotometer, Bio-Rad, Hercules, CA, USA). GI₅₀ was calculated for each healthy cell line.

The determination of TPC was carried out using the Folin-Ciocalteu method [48 (link)]. Gallic acid was used as a standard and was prepared at concentrations of 0.0, 2.5, 5, 10, 25, 50, 100, 250 and 500 mg/L to obtain a standard curve. For the reaction, 20 µL of distilled water, samples or standard solutions were added to the wells of a 96-well microplate. Then 100 µL of 1:10 diluted Folin-Ciocalteu was added to the wells. An incubation period of 4 min was necessary for the oxidation of the phenolic compounds by the Folin reagent. Afterwards, 80 µL of 7.5% sodium carbonate was added to inactivate the reaction. The microplate was incubated in the microplate reader for 45 min, and the absorbance was measured at 765 ƞm with an xMark Microplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada). TPC was expressed as mg gallic acid equivalent/g sample.

Total Flavonoid Content (TFC) was determined, according to Oomah, et al. [33 (link)]. The method consisted of mixing in a 96-well microtitration flat-bottom plate, 50 μL of the methanolic extract with 180 μL of distilled water, and 20 μL of a solution of 10 g/L 2-aminoethyldiphenylborate. The absorbance of the solution was measured with a spectrophotometer (xMark Microplate Spectrophotometer, BioRad, Osaka, Japan) at 404 nm after 15 min of reaction. Extract absorption was compared with a rutin standard (0 to 200 μg/mL). TFC was expressed as mg rutin equivalent per 100 g of dry matter (mg eq. rutin/g db).

Serum IgG antibodies specific to N proteins were measured with an ELISA. Briefly, high-binding 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with purified recombinant N proteins, 100 ng per well, in a carbonate–bicarbonate buffer (pH 7.4) overnight at 4 °C. Then, the plates were blocked with 1% BSA in PBS (pH 7.4) for 40 min at 37 °C and washed 3 times with PBS-T (PBS with 0.1% Tween 20). Serum samples were diluted 5-fold in PBS-T (1:500 to 1:121,500) and added to wells, followed by incubation for 1 h at 37 °C. Each sample was tested in duplicate. After washing, an HRP-conjugated goat antimouse IgG secondary antibody (Bio-Rad, Hercules, CA, USA) was added to each well and incubated at 37 °C for 1 h. Then, the plates were finally washed and developed with 1-Step TMB Substrate Solution (HEMA, Moscow, Russia) for 15 min. After the reaction with 1 M of H₂SO₄ was stopped, the resulting absorbance was measured at a wavelength of 450 nm (OD₄₅₀) using an xMark Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA). The area under the OD₄₅₀ curve (AUC) values were calculated as a trapezoidal square for each serum sample and expressed in arbitrary units.