Multiscan sky microplate spectrophotometer

The caspofungin concentrations for the biofilm MIC (sMIC) determination ranged from 0.5 to 32 mg/L, while the examined posaconazole concentrations ranged from 0.007 to 2 mg/L. The biofilms were washed three times with sterile physiological saline. After incubation at 37 °C for 24 h, the biofilms were washed with sterile physiological saline, and an XTT-assay was performed, as described previously [18 (link),49 (link),51 (link)]. The change (%) in metabolic activity was calculated based on absorbance (A_492nm) by using a Multiscan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), as:
The A_background corresponds to 100 µL drug-free and biofilm-free XTT-solution. The sMICs were defined as the lowest drug concentration resulting in at least a 50% metabolic activity decrease compared with the untreated control cells [18 (link),49 (link),51 (link)] and are presented as the median value of three independent experiments per isolate.

Lyophilized samples were ground to a fine powder with φ 5 mm steel ball in TissueLyser II (Qiagen, Venlo, Netherlands) for 2 min at 30 Hz. From each ground sample, three replicates were prepared and weighed separately. To prepare the protein extracts for measurement of the level of lipid peroxidation and the activities of GST, SOD and LAC, 1750 μL of cold extraction buffer (100 mM potassium phosphate buffer, with 0.1 mM EDTA, pH 7.0) was added to the ground lyophilized tissue, mixed with a micro pestle and vortexed briefly. The homogenate was centrifuged at 20000× g for 20 min at 4°C. Supernatant was collected and protein concentration was determined according to Bradford (1976) (link) using bovine serum albumin (Protein Standard, 2 mg/vial BSA, Sigma-Aldrich® Burlington, MA, United States) as a standard. The samples were aliquoted and stored at −20°C. All spectrophotometric measurements were done with a Multiscan Sky microplate spectrophotometer (Thermo Scientific™ ver 5.0) in 96-well plates (Nunc MaxiSorp™ flat-bottom, Thermo Scientific™).