Varioscan lux plate reader

The minimum inhibitory concentrations (MICs) of three antimicrobial agents: mitomycin C (BioShop, Burlington, ON, Canada), ciprofloxacin (MERC, Kenilworth, IL, USA), and rifampicin (BioShop, Burlington, ON, Canada) were determined for E. coli O157:H7 (ST2-8624) strain by using microdilution method described earlier [80 (link),81 (link)], with some modifications. Briefly, ciprofloxacin and rifampicin were dissolved in LB medium to the final concentrations ranging from 0.00005 µg/mL to 256 µg/mL and transferred to the wells of a 96-well polystyrene plates (Nest Scientific USA, Woodbridge, VA, USA). For mitomycin C, the solutions of concentrations from 0.0002 µg/mL to 125 µg/mL were prepared. In the next step, the mixtures were added to overnight bacterial cultures (inoculum of 3 × 10⁶ colony forming units, CFU). Plates were incubated with aeration at 37 °C in a shaking incubator (200 rpm; Benchmark Scientific, Sayreville, NJ, USA) for 24 h. Grow inhibition kinetics were determined with the Varioscan Lux plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 600 nm. The MIC was defined as the lowest concentration of tested agents that prevented a considerable growth of E. coli O157:H7 (ST2-8624) bacteria.

ScLPMO10D^CD, CjLPMO10A^CD, and SmLPMO10A were assessed for their peroxidase activity using a method described by Breslmayr et al.^{54 (link)}. This assay features a spectrophotometric method with 2,6-dimethoxyphenol (2,6-DMP) as a chromogenic substrate and H₂O₂ as co-substrate. The LPMO carries out a peroxidase-like reaction that converts 2,6-DMP into coerulignone, the formation of which can be measured spectrophotometrically at A₄₆₉. A solution containing 2,6-DMP and H₂O₂, and another solution containing the LPMO, were preincubated separately for 5 min at 30 °C in 50 mM sodium phosphate, pH 6.0. Reactions were initiated by mixing equal volumes of the two solutions, giving a final volume of 100 µL and a final concentration of 1 µM LPMO, 1 mM 2,6-DMP and 100 µM H₂O₂. Immediately after mixing, product formation was recorded in a Varioscan LUX plate reader (Thermo Fisher Scientific, Waltham, MA, USA), which was used to monitor the A₄₆₉ over a period of 10 min.

Sera of pregnant women who tested positive for HBV infection by PCR were each tested for the presence of HBcAb-IgG and HBcAb-IgM using IgG-HBcAb ELISA kit and IgM- HBcAb ELISA kit (MyBioSource Inc., San Diego, USA), respectively, following the manufacturer’s protocols. Absorbances were read at 450 nm using a Varioscan lux plate reader (Thermofisher Scientific, New Jersey, USA). Each sample was tested in duplicate, and those with standard deviations <15% were considered. The results obtained were interpreted in accordance with the manufacturer’s recommendations, and all samples tested were IgG positive and IgM negative.

ROS were detected using a 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) ROS Assay Kit from OZBiosciences (Marseille, France) according to the manufacturer’s instructions. Briefly, A549 and MRC5 cells were plated in 96-well black plates, optimised for plate readers, at 2000 cells/well and 4500 cells/well, respectively, and incubated for 24 h. Then the cells were exposed to IC25 concentrations of DK-164 or CC-78 for 24 and 48 h, and tert-Butyl hydroperoxide (TBHP) was used as a positive control. In preliminary experiments, the appropriate concentrations of TBHP for each cell line were determined as follows: 45 µM for A549 and 5 µM for MRC5. Afterwards, the cells were stained with 25 μM DCF-DA at 37 °C for 30 min in the dark. Subsequently, the extracellular dye was discarded, and the cells were rinsed with cold PBS twice. Positive controls were incubated with TBHP for 3 h before adding the fluorogenic dye DCF-DA. The experiment was repeated three times, each performed in triplicate. ROS levels were measured at Exc/Em 485/535 nm using a VarioscanLux Plate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescent intensity was analysed by SkanIt v.6.0.1 software (Thermo Fisher Scientific, Waltham, MA, USA).

TZM-bl cells were seeded into 96-well culture plates (100 μL of cell suspension or 10⁴ p cells per well) and placed in a CO₂ incubator. The test compounds were dissolved in DMSO at a concentration of 100 mM. After 24 h of incubation, various concentrations of the test compounds (1000, 500, 250, 125, 62.5, 31.25, and 15.6 μM) were added by titration to the TZM-bl cell culture, in triplicates. DMSO was added to the control wells at a concentration of no more than 1%. The plates with the introduced drugs were incubated in a CO₂ incubator at 37 °C under 5% CO₂. After 72 h of incubation, 20 μL of MTT working solution (5 mg/mL) was added to each well and the plates were again incubated under the same conditions. After 2 h, the plates were removed from the CO₂ incubator, and the medium in each well was replaced with DMSO solution (50 μL per well). The plates were shaken gently to dissolve the formazan crystals. The optical density of each well was determined at 570 nm using a Varioscan LUX plate reader (Thermo scientific). The survival rate of TZM-bl cells in the presence of the tested compounds was calculated using the formula: (OD of experimental wells—OD of medium)/(OD of control wells—OD of medium) × 100%, where OD is optical density.