NF-κB Reporter Gene Assay in THP1-Lucia™ Cells

BUT, DON, NX-3 and 10:1 and 1:1 combinations of BUT with the trichothecenes were prepared in reaction tubes to be further diluted 1:100 by the addition of the THP1-Lucia™ cell suspension. 10:1 and 1:1 ratios were selected due to the high variability of secondary metabolite concentrations in the available occurrence studies (8, 9, 34) and due to previously performed cell viability experiments in Woelflingseder et al. (12). Cellular concentration was determined by trypan blue exclusion, cells were centrifuged for 5 min at 250 × g and resuspended at a concentration of 1 × 10⁶ cells/mL in fresh, pre-warmed growth medium. Cell suspension was added to the respective toxin preparations into the reaction tubes. They were gently mixed and transferred as technical duplicates into a 96-well plate (100 μL/well). After 2 h of incubation at 37°C and 5% CO₂ in the humidified incubator, LPS (1 μL/well) at a final concentration of 10 ng/mL was added to the cells and incubated further for 18 h. 2% (v/v) water (LC-MS grade) with and without LPS treatment were used as solvent control. As recommended by the supplier, heat killed Listeria monocytogenes (HKLM) at a concentration of 20 × 10⁶ cells/well were applied as positive control for TLR activity. Following in total 20 h of incubation, well plate was centrifuged (250 × g, 5 min) and 10 μL/well of the supernatant were collected. Subsequently, the reporter gene activity assay (secreted luciferase) was performed according to the manufacturer's protocol. QUANTI-Luc™ solution (InvivoGen, US), containing the recommended coelenterazine substrate, was added for luminescence measurements.

In parallel cellular metabolic activity was monitored by the alamarBlue® assay (for details see 2.6). Therefore, after supernatant collection for the measurements of luciferase activity, 10 μL of alamarBlue® reagent (Thermo Fisher Scientific, Massachusetts, USA) were added to the cells and incubated for 2 h. Subsequently, after another centrifugation of the plate (250 × g, 5 min) 70 μL/well were transferred to a black 96-well plate and fluorescence intensity was measured at 530/560 nm (excitation/emission). Both luciferase activity and fluorescence intensity measurements were performed on a Synergy™ H1 hybrid multi-mode reader (BioTek, Bad Friedrichshall, Germany) assessing at least five independent experiments in technical duplicates. Modulation of NF-κB-dependent luciferase expression was referred to the respective THP1-Lucia™ cell viability determined as alamarBlue®-fluorescence intensity.