Infinite f200 microplate reader

For P. aeruginosa strains, the minimal inhibitory concentration (MIC) of C14R and the viability of the bacteria were determined similarly to the “Clinical and Laboratory Standards Institute” guidelines M27-A3 broth microdilution assay [59 ]. In short, 2.5 × 10³ bacterial cells were seeded in 200 µL Müller–Hinton broth in 96-well polystyrene microtiter plates (Sarstedt AG & Co. KG, Nümbrecht, Germany) and incubated at 37 °C with agitation at 900 rpm on an Eppendorf shaker. The effect of C14R on cell viability was tested in the presence of the peptides at different concentrations, and the cell viability was quantified by a resazurin reduction assay according to Patricia Bi Fai et al. [60 (link)]. The cells were incubated with 20 µL of 0.15 mg/mL resazurin solution for 2 h. During this time, the viable cells reduced resazurin to the fluorescent resorufin by the production of NADPH. Cell viability was then quantified by measuring the amount of produced resorufin by fluorescence measurements at an excitation wavelength of 535 nm and an emission of 595 nm using a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The curve was fitted using GraphPad PRISM 8 (Graphpad Software, Inc.; Boston, MA, USA) by a nonlinear fit using the Gompertz equation for MIC determination.

NQO1 enzymatic activity was measured using the NQO1 Activity Assay Kit (ab184867) from Abcam (Cambridge, UK). Briefly, the NQO1 activity assay is based on the dicoumarol-sensitive reduction of WST-1 in the presence of menadione using 10 ug of cellular lysate protein in a 96-well plate format. Progress of the reaction was measured at 1-min intervals by measuring absorbance at 450 nm on an Infinite F200 microplate reader (Tecan Group Ltd). A 10-min endpoint reading was chosen as a time point within the linear region of the reaction. Replicates were averaged, and activity was expressed as the OD_450nm following subtraction of OD_450nm + dicoumarol.

The PXR ligand‐binding assay of FKK5, FKK6, and FKK9 was performed using LanthaScreen TR‐FRET PXR (SXR) Competitive Binding Assay Kit (PV4839; Invitrogen, USA) according to the manufacturer's instructions. The assays were done with concentrations of tested compounds ranging from 1 nM to 25 μM. DMSO and 100 μM SR12813 were used as a negative and positive control, respectively. The reaction mixture was incubated at room temperature for 1 h in the dark, and then, fluorescent signals were measured at 495 and 520 nm, with the 340‐nm excitation filter, on Infinite F200 microplate reader (Tecan Group Ltd, Switzerland). Finally, the TR/FRET ratio was calculated by dividing the emission signal at 520 nm by that at 495 nm. All PXR‐binding assays were performed as two independent experiments, each with a minimum of four replicates. Final IC₅₀ was obtained by processing the data with GraphPad Prism 6 using standard curve interpolation (sigmoidal, 4PL, variable slope). SR12813 served as a positive control PXR agonist ligand, and it demonstrates an IC₅₀ of 0.127 μM, which is similar to previously published results (Shukla et al, 2009).

The antifungal effect of Pom-1A to Pom-1F on Candida spp. biofilm formation can be determined following the Clinical and Laboratory Standard Institute guidelines (M27-A3) [67 ]. For this, 2.5 × 10³ yeast cells were incubated in 200 µL of RPMI-1640 medium supplemented with L-glutamine, fluconazole, amphotericin B, and Pom-1A to Pom-1F. Incubation was performed on flat-bottomed polystyrene microplates with 96 wells (Sarstedt AG & Co. KG, Nümbrecht, Germany) for 24 h at 37 °C without shaking. The subsequent treatment with crystal violet was originally developed by George O’Toole for bacteria and adapted to Candida biofilms [68 (link)]. For this purpose, the planktonic phase was removed, and the wells were washed twice with 200 µL of demineralized water. The remaining biofilm cells were treated with 200 µL of 0.1% (w/v) crystal violet solution for 15 min. After removing the solution, they were washed again twice with 200 µL demineralized water, and the microtiter plates were dried for at least 24 h at 25 °C. The stain was dissolved with 200 µL of 30% acetic acid and transferred to a new plate after 15 min. The absorbance at 560 nm was measured using a Tecan Infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The resulting data were evaluated against the untreated controls so that the efficacy of the agents could be determined.

A resazurin assay was performed to detect cell viability. For this, 2 × 10⁴ cells per well were incubated for 48 h in a 96-well-plate in DMEM with supplements (100 µL) and different peptide concentrations (2.5 µg/mL, 20 µg/mL) at 37 °C containing 5% CO₂. Afterwards, 20 µL resazurin (0.15 mg/mL) was added to each well and was incubated for 24 h at 37 °C containing 5% CO₂. Thereafter, the fluorescence of resorufin was measured with a Tecan infinite F200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at an excitation wavelength of 535 nm and an emission wavelength of 595 nm.

A resazurin assay was used to detect the antifungal effect of Angicin on the viability of Candida cells. Therefore, 2.5 × 10³ cells were incubated in 200 μl RPMI-1640 medium with 300 mg/l L-glutamine and two different Angicin concentrations (25 μg/ml, 100 μg/ml) at 37°C for 24 h in a flat-bottomed polystyrene microtiter plate with 96 wells (Sarstedt AG & Co., KG, Nümbrecht, Germany) with shaking at 900 rpm on an Eppendorf shaker. For the following quantification of viable cells, a resazurin-Reduction-Assay was performed. In brief, 20 μl of 0.15 mg/ml resazurin (Sigma-Aldrich) solution was added per well and incubated for 2 h at 37°C while shaking at 900 rpm. Fluorescence measurement (excitation wavelength 535 nm, emission wavelength 595 nm) of the resulting resorufin (viable cells are able to reduce resazurin to resorufin) was then performed by using a Tecan infinite F200 microplate reader (Tecan Group). The resulting data were normalized to the untreated control and the efficacy of Angicin was determined.