Synergymx multi mode microplate reader

Biofilms were grown and treated as described above. Alternatively, biofilms were grown as described above but the treatment was performed in anaerobic conditions by placing the biofilm plates in a jar in the Anoxomat^TM Mark II System (Advanced Instruments INC, Massachusetts, USA). After 24 h of treatment at 37 °C, biofilm cells were washed and dissolved in 100 μL PBS by vigorously pipetting. Of this cell suspension, 10 μL was diluted and plated out on YPD agar plates to determine the number of fungal cfu after 2 days of incubation at 30 °C. To determine the amount of superoxide formed, the remaining 90 μL was stained by adding 10 μL of a 200 μM dihydroethidium (Life Technologies Europe) stock solution. This suspension was transferred to black-walled microplates (Greiner) and incubated for 20 min at room temperature in the dark. Finally, fluorescence was measured with a fluorescence spectrometer (Synergy Mx multi-mode microplate reader, BioTek, USA) at λ_ex 510 nm and λe_em 595 nm and values were corrected for fluorescence measured in blank wells. Cfu determination was also performed for the ΔΔΔsod4sod5sod6 mutant and the corresponding isogenic wild-type strain, CA-IF100, after treatment with 30 μM miconazole. Calculation of the Pearson correlation coefficient and analysis of significance was performed in GraphPad Prism 6.

The site-saturation mutagenesis library was screened as described4 (link)5 (link), with some modifications. Single colonies harboring the library mutants were grown in 1 mL LB medium supplemented with ampicillin in 96-well plates at 37 °C for 14 h. Cells were harvested by centrifugation (1,278 ×g, 10 min, 4 °C), then resuspended with 0.3 mL lysis buffer (50 mM Tris-HCl, 10 mg/mL lysozyme, pH 8.0) and incubated at 37 °C for 60 min. The cell debris were removed by centrifugation (1,840 ×g, 10 min, 4 °C) and the crude enzyme extracts were used for downstream enzymatic reactions. Enzyme assays were carried out by incubating 50 μL of crude enzyme extracts with 50 μL of the substrate solution (100 mM potassium phosphate buffer, 0.2 mM 4-MU, 200 mM sucrose, pH 6.0) in 96-well plates at 37 ^oC for 5 h. Fluorescence of each well (excitation at 350 nm and emission at 460 nm wavelength) was determined both before and after the incubation with a SynergyMx Multi-Mode Microplate Reader (BioTek, Vermont, USA). The fluorescence differences of the variants between 0 and 5 h were calculated and the mutants with higher fluorescence decrease than the wild-type enzyme were selected for rescreening. The selected mutants were re-cultured in LB and the crude enzyme extracts were used to react with 4-MU and sucrose, and the productions of 4-MUG were determined with HPLC as described below.

β-Galactosidase activity was assayed as described by Miller [44 (link)], with some modifications. The reaction mixture was prepared by addition of 650 μL buffer Z, 40 μL of 12 mM 2-nitrophenyl-β-d-galactopyranoside (oNPG) and 10 μL of the cell lysate. After incubation at 37 °C for 10 min, the reaction was terminated by the addition of 185 μL of 1 M sodium carbonate. The absorbance at 420 nm was measured with a SynergyMx Multi-Mode Microplate Reader (BioTek, Vermont, USA). β-Galactosidase activity in Miller units was calculated as (1000 × OD₄₂₀)/(T × V × OD₆₀₀), where T represents the reaction time (minute) and V represents the volume of cell lysate used in the assay (mL).

Optical density measurements were collected using a Synergy Mx Multi-mode Microplate Reader (Biotek). Overnight grown cultures were diluted 1:1000 in LB and further grown in Greiner 96-well plates for 2 h. Then, cefsulodin was added to a final concentration of 100 µg/ml in the wells and the OD was monitored at regular time intervals. OD was measured at 595 nm.

A quantitative cell viability analysis was performed using the MTT test [52 (link),53 (link),55 (link)].
NCTC clone 929 cells were grown and treated with CeO₂NPs for 24 h as described above (see Section 4.11.2). For other examined culture cell lines, the cells were seeded in 96-well plates (5 × 10³ cells per well) and grown overnight. Then the cells were exposed to 8–600 µg∙mL⁻¹ solution of CeO₂NPs-5FU and incubated for 72 h.
After the incubation period, the culture medium was replaced with a serum-free medium containing the MTT reagent (5 mg∙mL⁻¹, Alfa Aesar, UK). After 2 h, the medium was removed, and the formed formazan crystals were dissolved in DMSO. The optical density (E) of the solution was measured at 570 nm and 620 nm wavelengths using a Synergy Mxmulti-mode microplate reader (BioTek Instruments, Winooski, VT, USA). The proportion of viable cells (Nv) was calculated according to Formula (2):
Triplicate wells were set up in each plate and three independent experiments were performed to determine the IC₅₀ concentration.

The activity assay of TYO was performed at 37 °C by monitoring the absorbance increase at 240 nm which indicates the formation of H₂O₂^{47 (link)}. A reaction mixture (200 μL) for the assay contained 100 mM phosphate buffer (pH 7.0), 0.75 mM dopamine and 0.002 mg mL⁻¹ purified TYO protein. Reaction was performed at 37 °C for 10 min. The H₂O₂ formation was monitored at 240 nm, with a SynergyMx Multi-Mode Microplate Reader (BioTek, USA). One unit of activity was defined as the amount of enzyme that catalyzes the production of 1 μM of H₂O₂ per min.
The HpaBC activity was assayed by quantifying the product formation with HPLC. One unit of activity was defined as the amount of enzyme catalyzing the conversion of 1 mM tyrosine per minute under the assay conditions^{19 (link)}.