Resazurin solution

Biofilm cells were prepared and incubated with vB_EfaS-271 lysate as described in Section 4.11. The resazurin assay was conducted according to the protocol described elsewhere [82 (link)]. First, the liquid medium was removed from each well. Next, the surface-attached cells were suspended in 1 mL of PBS. Then, resazurin solution (Sigma-Aldrich) was added to each well to a final concentration of 24 μg/mL. The plates were gently shaken and incubated in an EnSpire multimode plate reader for 50 min at room temperature. The fluorescence of the produced resorufin (λexc = 570 nm and λem = 590) was measured every 5 min. The results are presented as fluorescent units (FU). The procedure was repeated three times.

Clonogenic survival was evaluated using the colony forming assay described previously^{35 (link)}. The resazurin assay was used to measure 2D growth curves over one week. On day 0, 200 μl of cell suspension containing 2000 (treated) cells were plated in flat bottom 96-well plates. For each condition, six replicate wells and a total of six well-plates were used. Plates were incubated at 37 °C in a CO₂ incubator and one was removed every 24 h for viability testing. The plate was subjected to a resazurin assay, as follows: 100 μl of culture medium per well was removed, and replaced with 20 μl resazurin solution (0.15 mg/ml (Sigma Aldrich Ltd.) in Dulbecco’s PBS, filter sterilized through a 0.2 μm membrane filter). After incubation for 4 h at 37 °C the fluorescent signal at an excitation/emission wavelength of 560/590 nm was measured using a multi-plate spectrophotometer (FluoStar Omega, BMG Labtech). Readings were normalized to the signals measured in culture medium with resazurin alone. The assay was repeated at least three times for each condition ( $n\ge 3$ ). Mean values and standard deviations are reported.

The optimized Blastocystis viability assay was used to measure 50% inhibitory concentrations (IC50s) of different drugs for the Blastocystis strains [49] (link). Briefly, stock solutions of drugs were prepared in dimethyl sulfoxide (DMSO), diluted in pre-reduced Blastocystis culture medium, transferred to 96-well plates and pre-reduced in anaerobic jar for 4 h at 37°C. Parasites were counted and 0.5×10⁶ cells were incubated in each well of a standard 96-well plate with dilutions of different drugs ranging between 0 and 100 µg/ml. The drug concentration range was reduced or increased depending on the results of the first test. A range of parasite counts between zero and 0.5×10⁶ per well were used for viability control. The final DMSO concentration was kept constant at 0.5% in each well. Since the parasite redox activity varies with volume, the final total volume of each well was kept constant at 200 µl. After 24 h of drug exposure, resazurin solution (Sigma) was added to each well at a final concentration of 10% (v/v); 3 h after incubation, under anaerobic conditions at 37°C, fluorescence readings of resazurin were taken at 550-nm excitation and 580-nm emission wave lengths using a Tecan Infinite M200 reader. Values were imported into GraphPad Prism5 software and IC50s were calculated.

The assay followed the protocol of Riss et al. [34 ], with some modifications. Briefly, splenocytes were transferred into 96-well plates (1.1 × 10⁶ cells/mL), and 10 µL resazurin solution (Sigma, St. Luis, MO, USA) was added to each well. The plate was then incubated (37 °C, 24 h, 85 HR, and 5% CO₂). To quantify the viability after the incubation period, a fluorescence plate reader was used at 530 nm excitation and 590 nm emission (Varioskan™ Flash Multimode Reader, Thermo Scientific). A 10% (v/v) DMSO solution plus the cells was used as the cytotoxic control, and water plus the cells as the positive control. The percentage of cell viability was calculated as described in Equation (15): $$\% of cell viability=\frac{F_{mat}-F_{blank}}{F_{control}-F_{blank}}\ast 100$$
where F_mat is the fluorescence of the cells in the presence of material, F_blank is the fluorescence of wells in the absence of the cells, and F_control is the fluorescence of cells without the material.

Cell viability to determine the half maximal inhibitory concentration (IC50) values and synergistic action of test drugs was measured by resazurin reduction assay as previously described [46 ]. Briefly, test drugs were diluted serially in 96-well plates and cells were seeded at 2.5 × 10⁴ cells/mL. Plates were incubated at 37 °C in 5% CO₂ for 72 h (h), after which resazurin solution (Sigma-Aldrich, Dorset, UK, cat. 62758-13-8) was added; plates were then incubated for a further 4 h. A SpectraMax M5 Plate Reader (Molecular Devices, California, USA) was used for measuring fluorescence. IC50 values were determined using GraphPad 8 (GraphPad, California, USA) and combination indices (CI) were calculated using CompuSyn (ComboSyn Inc., New Jersey, USA) software according to the Chou-Talalay method [47 ]. A CI of ‘>1’ indicates an antagonistic, ‘1’ additive and ‘<1’ synergistic interaction.

Cell vitality was determined by resazurin assay according to the method of Mehring et al. [27 ]. For this purpose, the cell suspension was diluted to an OD of 0.2 with Sorensen buffer (pH 7.4). For each 20 μl of cell suspension, another 180 μl of Sorensen buffer and 20 μl of a resazurin solution (2 mg/ml) (Sigma Aldrich) were added to a 96 well plate. Fluorescence was measured at 540 nm excitation and 590 nm emission (2030 Multilable Reader VICTOR X3, PerkinElmer, USA). Calibration was performed using standards that contained 0%–100% live cells of S. pasteurii.

Cells were seeded into the 96-well tissue culture plates and, on the other day, compounds were added in different concentrations in duplicate for 72 h. Upon treatment, the resazurin solution (Sigma Aldrich, St. Louis, MI, USA) was added for 4 h, and then the fluorescence of resorufin was measured at 544 nm/590 nm (excitation/emission) using a Fluoroskan Ascent microplate reader (Labsystems, Budapest, Hungary). The GI₅₀ value was calculated from the dose–response curves that resulted from the measurements using GraphPad Prism 5.