Multiskan fc spectrometer

Tissue injury was assessed by the release of lactate dehydrogenase (LDH), which converts pyruvate to lactate, into the incubation medium at the end of the 120-min reoxygenation period. The absorbance was measured at a wavelength of 340 nm using a MultiSkan FC spectrometer (Thermo Fisher Scientific, Waltham, MA). The results were expressed as arbitrary units (AU)/g tissue wet wt.
The viability of the muscle tissue was assessed at the end of the 120-min reoxygenation period by the reduction of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, St. Louis, MO) to a blue formazan product, the absorbance of which was measured at 550 nm using a MultiSkan FC spectrometer (Thermo Fisher Scientific, Waltham, MA). The results were expressed as AU/g wet wt.

The bioavailability of NO was assessed using a colorimetric assay kit (Cayman, Ann Arbor, MI) performed according to the manufacturer’s instructions. The samples were incubated with nitrate reductase, which converts nitrates to nitrites. The latter react with the Griess reagent to yield a colored azo dye product, the absorbance of which was measured at 540 nm using a MultiSkan FC spectrometer (Thermo Fisher Scientific Waltham, MA). The results were expressed as μM NO/mg protein.

All strains were subcultured twice from frozen stocks on Sabouraud dextrose agar slants supplemented with chloramphenicol and gentamycin (Bio-Rad Laboratories, Feldkirchen, Germany) at 35 °C and 95% humidity. Incubation time was 24 h for Candida spp. and 7 days for filamentous fungi in accordance with EUCAST recommendations for slow growing molds [33 ]. Suspensions were counted in a hemocytometer and adjusted to the final inoculum size of 2 × 10⁵ colony forming units (CFU)/mL in water for yeasts, and water containing 0.1% (v/v) of Tween 80 for molds, which should prevent fungal growth on surfaces of the wells [35 (link)]. After the distribution of 100 µL of the final inoculum into each well, microplates were incubated at 35 °C, with 95% humidity. Incubation time was 24 h for Candida and Rhizopus species and 48 h for Aspergillus species. After incubation optical densities were read spectrophotometrically at a wavelength of 530 nm using a MultiSkan FC spectrometer (Thermo Fisher Scientific). Before the reading, microplates containing yeast inocula were shaken for 2 min at 1100 rpm with a PMS-1000 Microplate Shaker (Grant Instruments, Shepreth, UK). All experiments were run in duplicate.

The enzymatic activity was measured with the ABTS assay according to Majcherczyk et al. (1999 (link)). The activity (U), was defined as the amount of the enzyme catalyzing the oxidation of one µmol of ABTS per minute. The pH was adjusted to 4.5 using McIlvaine buffer. The measurements were performed using a Multiskan FC Spectrometer from Thermo Scientific with 96-well plates. For each measurement, V_s = 50 µl of the sample was used, and its pH adjusted to 4.5 by adding 150 µl of the McIlvaine buffer. Then, 50 µl of 0.5 mM ABTS solution was added. Therefore, the volume of every measured sample was equal V_tot = 250 µl, and its optical length in the single well was equal d = 0.63 cm. The measurements of absorbance change ΔE were performed in 37 °C at 420 nm wavelength over Δt = 3 min. The extinction coefficient for ABTS at 420 nm wavelength is equal ε₄₂₀ = 0.04321 L mmol⁻¹ cm⁻¹ (Prinz et al. 2012 ). If the sample’s activity was too high for the spectrometer’s range, it was diluted with a dilution factor D. All activity measurements were performed in triplicates. To calculate the enzymatic activity, the following equation was used: $$\text{act}\left[U·L^{-1}\right]=\frac{\mathrm{\Delta }E·D·V_{\text{tot}}}{\mathrm{\Delta }t·V_{\text{s}}·d·{\mathit{\epsilon }}_{420}}$$