Xmark microplate spectrophotometer

The catalase activity assay is based on the measurement of the hydrogen peroxide substrate produced by the action of catalase (CAT). Briefly, 50 µg of epidydimal protein homogenate was diluted in the assay buffer (50 mM potassium phosphate buffer, pH 7.0). The reaction was started by the addition of the samples, the assay buffer and 10 mM of H₂O₂ solution. This mixture was left to react for 5 min; the reaction was stopped by adding 15 mM sodium azide solution and transferred to a new well. Then, the color reagent (150 mM potassium phosphate buffer, pH 7.0; 0.25 mM 4-aminoantipyrine; 2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid) was added and the mixture left to stand at room temperature for 15 min. The assay was performed in a 96-well microplate and the enzymatic activity was measured at 520 nm in a spectrophotometer (Bio-Rad xMARK^TM, Microplate Spectrophotometer, Hercules, CA, USA). The activity of CAT was expressed as activity µmol/min/mL, in which one unit of CAT activity corresponds to the amount of the enzyme that decomposes 1 μmol of H₂O₂ in O₂ and H₂O per min. at pH 7.0 at 25 °C.

Bacteria were grown in N-minimal medium with 10 mM MgCl₂ at pH 7.5 to saturation. One milliliter of bacterial cells were collected, washed twice with medium not supplemented with MgCl₂, and diluted 1:100 into wells of a 24-well plate containing 1 ml of medium with 10 μM or 1 mM MgCl₂ at pH 7.5 or pH 5.5. The C18G peptide was added to medium at 5 μg/ml. A plate was covered with a Breathe-Easy sealing membrane (Sigma) to prevent evaporation. By using an xMark^TM Microplate Spectrophotometer (Bio-Rad), bacteria were cultivated at 37°C with shaking, and the OD₆₀₀ values were measured every 5 min up to 8 h.

A neutral red uptake assay was performed to determine cell viability and to confirm the visual perception of protection from CPE²¹ . Following the visual evaluation of results after 24 h of incubation, the contents from each well were removed and a 100 µl aliquot of 40 µg/ml neutral red solution was added. Plates were incubated for 3 h at 37 °C with 5% CO_2, then washed with 150 µl of phosphate buffered saline (PBS pH 7.3). To elute the stain, 150 µl of neutral red destain solution was added to each well and the plates were placed on a shaker at 120 rpm for 10 min. The optical density of each well was measured at 540 nm using an xMark^TM microplate spectrophotometer (Bio-Rad). Wells without cells served as blanks. The percentage of viable cells in wells containing treated and untreated culture filtrates was calculated relative to the untreated control cells, which were assumed to be 100% viable.

MDCK cells were transfected with ASOs in concentration from 1 uM to 4 uM and seeded on 96-well plate as for experiments of antiviral tests described above. After transfection cells were incubated at 37 °C in air with 5% CO₂ and after 12 h medium was changed for fresh one followed by incubation of another 6 h. MDCK cells treated with LPF-2000 were also evaluated. The cytotoxicity of ASOs were evaluated using 1% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazolium). MDCK cells were incubated for 2 h with tetrazolium at 37 °C in air with 5% CO₂ and after 2 h, solution was changed for DMSO (100 ul/well) and incubated for 15 min. in dark. The rate of formazan formation was determined by measuring the absorbance (570–650 nm) on xMark^TM Microplate Spectrophotometer (BioRad). Cells enzymes are capable of reducing the tetrazolium to its insoluble formazan. The 570–650 nm reading value is directly proportional to the number of living cells. Cytotoxicity assays were normalized to viability of cells treated only with Lipofectamine 2000. The mean and standard deviation were calculated with Microsoft Excel software from three independent experiments each containing three technical repeats.

A cell counting kit (CCK-8/WST-8) (ab228554, Abcam, Waltham, MA, USA) based on monosodium salt WST-8 was used to count the number of viable cells in accordance with the manufacturer’s instructions Each sample was transferred in a volume of 100 mL to a 96-well plate, and 10 mL of the dye was then applied to each well. After 3 h of incubation at 37 °C in the dark, cellular dehydrogenases degraded WST-8 to an orange formazan product that was soluble in the buffer. The WST-8 kit is a colorimetric assay in which salt is reduced by metabolically active cells, to form an orange formazan product that is quantifiable by plate reader spectrophotometry. It is a direct assay for cell viability and proliferation, indicated by the color intensity. The absorbance of the cell suspension was determined using a microplate reader at 450 nm (Bio-Rad xMark^TM Microplate Spectrophotometer; Hercules, CA, USA). The quantity of formazan produced was directly correlated with the number of live cells [37 (link)].

The assay of Glutathione Reductase activity was based on the reduction of oxidized glutathione (GSSG) and posterior reconversion to reduced glutathione (GSH) by Glutathione Reductase, using one molecule of NADPH. Briefly, 100 µg of epidydimal protein homogenate was diluted in a glutathione reductase assay buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA) and 1 mg/mL BSA. Then, this mixture was incubated for 10 min. at 25 °C in a reaction buffer containing 2 mM oxidized glutathione solution, the glutathione reductase assay buffer and 3 mM of 5,5-dithiobis (2-nitrobenzoic-acid). The reaction was started by the addition of the 2 mM NADPH. The assay was performed in a 96-well microplate and the enzymatic activity was measured following the increase of absorbance at 412 nm using a spectrophotometer (Bio-Rad xMARK^TM, Microplate Spectrophotometer, Hercules, CA, USA). The activity of glutathione reductase was calculated by the mean of the slopes, obtained at 0 s and at 88 s, using a molar extinction coefficient of 14.15 mM⁻¹·cm⁻¹, and was expressed as Units/mg.