Powerwavex

GPx, GRd and CAT constitute the first line of antioxidant defense of our body. Their activities were determined in the erythrocyte fraction. GPx activity was spectrophotometrically measured in a 96-well plate (PowerWaveX; Bio-Tek Instruments, Inc., Winoosky, VT, USA). The method consisted of an indirect measurement of NADPH consumption. GRd activity was also measured spectrophotometrically, using a commercial kit (703202; Cayman chemical, Ann Arbor, MI, USA). Oxidation of NADPH to NADP+ resulted in a decrease in absorbance at 340 nm, which was directly proportional to GRd activity.
CAT activity was measured by the rate of H₂O₂ decomposition, according to the method of Aebi [27 (link)]. All enzyme activity was expressed as µmol/min/g Hb. Hemoglobin concentration was spectrophotometrically determined by Drabkin’s method [28 (link)].

Increasing concentrations of 5-FU and ITC were added to cells in the logarithmic phase of growth, next the incubation lasted 24 or 72 h. The cell growth was evaluated using the MTT (3,-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide) assay. At the end of the incubation, the medium was removed and the 0.25 mg/mL MTT (Sigma Aldrich) was added. The absorbance of formazan was measured at a wavelength of 570 nm with background subtraction at 690 nm. The microplate scanning spectrophotometer (PowerWave X, BioTek, Winooski, VT, USA) was used for the measurements.

The presence of each of the secreted NTFs was quantified using ELISA specific kits (RayBiotech, Norcross, GA, United States). The assays were conducted according to the manufacturer’s protocols in duplicate, and absorbance was read at 450 nm using an ELISA reader (PowerWave X; BioTek Instruments, Winooski, VT, United States).

The murine monocyte/macrophage cell line P388/D1 and murine macrophage-like cell line RAW264.7 were grown in a 96-well plate in DMEM supplemented with 5% (v/v) FBS. Prior to the experiment, the medium was replaced with fresh DMEM enriched with 2% (v/v) of FBS (negative control) or supplemented with 1) 100 ng/ml lipopolysaccharide (LPS, Sigma), 2) 10 ng/ml IFN (R&D Systems), 3) 0.01–1000 nM BacSp222, or 4) BacSp222 combined with LPS or IFN . The cells were then cultured for another 24 h to induce iNOS expression and nitric oxide synthesis. The nitrite levels were measured in culture medium by a nitrate assay performed on a microplate. One hundred microliters of cultured media was incubated with 100 μl of Griess reagent (1% (w/v) sulphanilic acid/0.1% (w/v) N-(1-naphtyl) ethylenediaminedihydrochloride (Sigma) in 2.5% (v/v) H₃PO₄) at room temperature for 10 min. Then, the absorbance was measured at 545 nm using a microplate reader (PowerWave X, BioTek Instruments). Before use, the solution of BacSp222 was tested for LPS contamination using an E-TOXATE assay kit (Sigma). The measurements ascertained that the level of LPS in 1 μM bacteriocin solution was equal to or less than 3 pg/ml. As verified in a control experiment (data not shown), such a concentration did not influenced iNOS activity in the cells used.

The SRB binding assay is based on the pH-dependent binding of SRB to basic amino acids of cellular proteins under mild acidic conditions. The colorimetric evaluation of bounded SRB thus provides an estimation of the total protein mass, which is related to the number of cells in culture [49 (link)]. Twenty-four hours after exposure to the compounds, the cell culture medium was removed, the cells were washed with HBSS (+/+) and fixed overnight, at −20 °C, with a methanolic solution of 1% acetic acid (v/v). The fixation medium was then removed, and the cells incubated with a 0.05% SRB solution (prepared in 1% acetic acid) for 60 min, at 37 °C. After incubation, the SRB solution was removed and the cells washed with 1% acetic acid (v/v) to remove the unbound dye. The bounded SRB was then extracted with a Tris base solution (10 mM, pH 10.5) and the absorbance was measured, at 540 nm, in a multi-well plate reader (PowerWaveX BioTek Instruments, VT, USA). The percentage of SRB binding relatively to that of the control cells (0 µM) was used as the cytotoxicity measure. Six independent experiments were performed, in triplicate.

The viability of the cells in the neuronal cultures was assessed by their ability to uptake thiazolyl blue tetrazolium bromide (MTT). The cells were incubated with MTT for 1 h, then lysed with dimethyl sulfoxide (DMSO) and left at room temperature in the dark overnight. The lysates were then read on a plate reader (PowerWave X, Bio-Tek) at the absorbance wavelength of 540 nm.