Wallac victor 1420

Working reagent (WR) used in Pierce^TM quantitative peroxide assay reagent was prepared in accordance to manufacturer’s recommendation (Thermo Fisher Scientific, USA). Overnight cultures of S. aureus JE2 strain were adjusted to optical density 0.5 McFarland standard, then the photosensitiser (RB) was added followed with 15 min incubation at room temperature, and afterwards, irradiated with sub-lethal dose of aPDI or aBL. Next, 10 µL of WR were added to 100 µL of irradiated sample and the spectrophotometric measurement was performed at the wavelength 570 nm (Wallac 1420 Victor, Perkin Elmer). Additionally, the same experiment was performed for cells treated with aPDI (without photosensitizer), cells treated only with RB and cells alone. For estimation the H₂O₂concentration in tested samples, the standard curve was prepared.

The NOs/w levels were determined indirectly by measuring NO₂¯ and nitrate (NO₃¯) levels as NO end products using a commercial kit (Nitrate/Nitrite Colorimetric Assay Kit, Cayman Chemical IN 780001) and following the manufacturer’s instructions. The NO₂¯/NO₃¯ levels were determined in two steps. In the first step, NO₃¯ is converted to NO₂¯ by adding the enzyme nitrate reductase. In the second step, NO₂¯ is converted to the azo compound by adding the Griess reagent. The concentration of NO₂¯ is determined by measuring the absorbance of the formed dark purple azo compound. The absorbance was read at 540 nm in an automatic microtiter plate reader (Perkin Elmer, Wallac 1420 Victor). The NO₂¯ levels are expressed in μM. To normalize NOw with TGw as the mass equivalent of the sampled thyroid tissue, we defined the TGw/NOw indices for each subject in the study.

In order to determine the in vitro antioxidant capacities of both ExF and ExMR, the ORAC method [26 (link)] was employed. Also, a comparison between ORAC unprocessed (ExMR) and processed extract by ICH (ExMR ICH) was performed.
The fluorescence probe fluorescein (FL, 10 nM) was used as a reference compound attacked from peroxyl free radicals that are generated from APPH (100 mM) solution. In order to calculate the area under a curve (AUC) of the tested compounds (12.5 μg/ml), the reaction was following at 37°C (pH 7.0) until a fluorescence decay of FL solution in the presence of APPH. Each measurement was repeated at least three times, using a Wallac 1420 Victor 96-well plate reader (PerkinElmer, USA) with a fluorescence filter (excitation 485 nm, emission 520 nm). The Trolox (12.5 μM) was used as an antioxidant control.
The ORAC value refers to the net protection area under the quenching curve of fluorescein in the presence of an antioxidant. The final results (ORAC value) were calculated and expressed in ORAC units (Trolox micromol per microgram of sample (μmol/μg)).
$\begin{array}{c}\mathrm{O}\mathrm{R}\mathrm{A}\mathrm{C}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\left(\mu \mathrm{m}\mathrm{o}\mathrm{l}/\mu \mathrm{g}\mathrm{r}\mathrm{a}\mathrm{m}\right)=\frac{K\left(S\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}–S\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)}{\left(S\mathrm{T}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{x}–S\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\right)},\end{array}$ where K is a sample dilution factor and S is the area under the fluorescence decay curve of the sample, Trolox or blank, calculated with Origin®7 (OriginLab Corporation, Northampton, USA).

Liquid assays were performed at room temperature in a clear-bottom 96-well plate by using starting cultures with an optical density at 595 nm of 0.15. The bacteria were treated photodynamically or with ciprofloxacin, and the luminescence was recorded every 2 h for 6 h in a Wallac 1420 Victor multilabel counter (Perkin-Elmer). Each determination was replicated three times.

For quantitative assessment of cell viability three different BG concentrations, being 1, 2.5, and 5 mg/mL, were used to evaluate whether BG passivation has influence in a dose-dependent manner.
After removing CCM and washing cells in 100 µL PBS, 10 µg/mL FDA staining solution was added to the BMSCs. Culture plates were incubated for 5 min at 37 °C. Cells were washed in 100 µL PBS and thereafter lysed in 150 µL 0.5% Triton X-100 (Sigma-Aldrich) for 5 min. In a Wallac 1420 Victor microplate reader (Perkin Elmer, Waltham, MA, USA) fluorescence was detected at 535 nm.