Varioscan lux

After detachment, 2.5 x 10³ T24 cells and 12.5 x 10³ SV-HUC-1 cells were seeded per well in a 96-well plate. The following day, different concentrations of moxifloxacin, ofloxacin, norfloxacin, or enrofloxacin were added (25, 50, 100, 200, 500, and 800 μg/mL), and after that, the cells were incubated for 24 and 48 hours at 37°C in 5% CO₂. Untreated cells (with the acidified medium used for dissolving the drugs) were used as a control. After the incubation, 100 μL of 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) reagent (1 mg/mL) was added to each well, and the plate was placed in the dark for two hours at 37°C. Next, the supernatant was removed, and dimethyl sulfoxide (DMSO, POCH, Poland) was added to dissolve formazan crystals. The absorbance was measured spectrophotometrically at 570 nm and 655 nm using a Varioscan LUX plate reader (ThermoFisher Scientific, USA). Obtained absorbance results were used to calculate cell viability after fluoroquinolone treatment and to calculate the lethal concentrations (LC) causing the death of 10, 50, and 90% of cells.

A cell viability assay was performed using a previously published method [22 (link)]. Dr. Torsten Nielson, University of British Columbia, provided the human Yamato-SS cell line (CVCL_6C44)which was maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO₂. The in vitro cell viability assay was performed using MTT (3-(4, 5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent to compare the viability and anti-proliferation effects of jaspine B with jaspine B liposome in different concentrations. Cell viability was normalized using a drug-free medium or empty liposome as a control treatment. Cells were seeded in a 96-well tissue culture dish at 10,000 cells/well and incubated for twenty-four hours. Serial dilutions of jaspine B and jaspine B liposomes were prepared so that the final concentration ranged from 0.01 to 100 µM. After 72 h of treatment, 5 mg/mL MTT was added to each well, resulting in a final concentration of 0.5 mg/mL MTT; cells were incubated at 37 °C and 5% CO₂ for 2.5 h. Formazan crystals were resolubilized in 10% SDS and 0.01 M HCl, and the absorbance of the samples was measured at 570 nm and normalized to 650 nm using a microplate reading spectrophotometer, VarioscanLux (Thermofisher, Waltham, MA, USA). Each experiment was performed in triplicate.

The growth rate of phage-resistant mutants (as well as control strains) was estimated by measuring optical density (OD₆₀₀) kinetics with the starting density of 10⁶  CFU/ml. Cultures prepared in 24-well plates were incubated while shaking for 18 h at 37 °C in a microplate reader (Varioscan Lux, Thermo Scientific). The OD value (λ = 600 nm) was measured at 20-minute intervals. The results of the growth rate measurements were expressed as the cumulated OD values. Each experiment was performed in triplicate. The data was analyzed using the OriginPro 8.5 (OriginLab Corporation, Northampton, Massachusetts, USA). All values were expressed as mean ± SD and significant differences between variations (denoted p < 0.001) were assessed with the Tukey test using one-way ANOVA.

The total phenolic content was determined through the Folin–Ciocalteau reagent [33 (link)]. Briefly, a solution of the test extracts (20 μL), 10% Folin–Ciocalteau reagent (40 μL), and 7.35% sodium carbonate (140 μL) were added to a well of a 96-well plate. The blank comprised deionized water (20 μL), 10% Folin–Ciocalteau reagent (40 μL), and 7.35% sodium carbonate (140 μL). The mixture reacted in the dark for 1 h, and then absorbance was measured at 765 nm using a Thermo Scientific Varioscan LUX microplate reader (Thermo Fischer Scientific, Waltham, MA, USA). The absorbance values were converted to total phenolic content using a calibration curve previously constructed with a gallic acid standard (Sigma-Aldrich, St. Louis, MO, USA). Thus, the total phenolic contents were expressed as mg gallic acid equivalents per gram of seed powder (mg eq gallic acid/100 g dsp). Determinations were performed by using three biological and three technical replicates. The analytical method performance was monitored for each analysis batch, involving five QC samples spiked with 10% gallic acid to assess the method response variations (CV < 5%).

The ability to form biofilm was tested on 96-well, flat-bottomed, sterile polystyrene plates (Promed^®) according to Stepanović et al. [22 (link)], as described previously [19 (link),23 (link)]. Absorbance at 570 nm wavelength was measured with spectrophotometric microplate reader Varioscan LUX (Thermo Scientific, Waltham, MA, USA). Wells containing broth only were used as negative control. Optical densities (ODs) for each test strain were determined from the arithmetic mean of 3 replicates. The value obtained was compared with the cut-off value (ODc). ODc is defined as three standard deviations above the mean OD of the negative control. Based on the results, the isolates were classified as: non-biofilm producers (OD ≤ ODc); weak biofilm producers (ODc < OD ≤ 2 × ODc); moderate biofilm producers (2 × ODc < OD ≤ 4 × ODc); and strong biofilm producers (4 × ODc < OD).

The Toll-like receptors (TLRs) stimulation of monocytes, major forms of innate immune sensors, was assessed according to manufacturer protocol using THP1-XBlue™ cell line (InvivoGen, Toulouse France). THP1-XBlue™ cells derive from the human monocytic THP-1 cell line and express an NF-κB- and AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon TLR2, TLR1/2, TLR2/6, TLR4, TLR5 and TLR8 stimulation, THP1-XBlue™ cells activate transcription factors and subsequently the secretion of SEAP which is easily detectable when using QUANTI-Blue™, a medium that turns purple/blue in the presence of SEAP. The results were analyzed using a microplate reader (Varioscan Lux, Thermo Scientific).

For treatment studies, organoids were initially plated as triplicates in 96-well plates (with 50 µL Matrigel and a 150 µL medium) or 384-well plates (with 15 µL Matrigel and a 50 µL medium). The cell viability of treated PDOs was analyzed after seven days by using a Presto Blue Cell Viability Reagent (Invitrogen). The Presto Blue reagent was mixed with a human colon medium in a ratio of 1:10 according to the manual, and PDOs were overlaid by the mixture. After incubation for 3 h at 37 °C, the fluorescence was measured (Varioscan Lux, Thermo Scientific). Relative viability was normalized to the respective untreated control per PDO. The areas under the curve (AUCs) per drug response curve were determined by GraphPad Prism (version 9.3.1, GraphPad, San Diego, CA, USA) and normalized to the expected AUC if the viability would be constant at 100%.