Development and Characterisation of a Novel NF-κB Reporter Cell Line for Investigation of Neuroinflammation

Figure 2

GFP and luciferase expression is increased in U251-NFκB-GFP-Luc cells after activation of NF-κB signalling. (a) Stimulation with 10 ng/ml TNFα for 24 h significantly increases NF-κB-dependent luciferase activity in U251-NF-κB-GFP-Luc cells. Mean ± SEM from three independent experiments are shown, compared using Student’s t-test (unpaired, two-tailed, CI 95%). . (b) Fluorescence microscopy and (c) flow cytometry was applied to visualize the increase of GFP expression in U251-NF-κB-GFP-Luc cells after 24 h stimulation with 10 ng/ml TNFα. Scale bar: 100 μm. (d) U251-NF-κB-GFP-Luc cells were triggered for 24 h with different concentrations of LPS derived from E. coli (LPS_{E. coli,} blue) or S. minnesota (LPS_{S. minnesota,} yellow) (0.01, 0.1, 1, 10, and 100 μg/ml), and NF-κB-dependent luciferase activity was analysed. A significantly higher luciferase activity was shown using E. coli LPS compared to S. minnesota LPS. (e) U251-NF-κB-GFP-Luc cells were treated with different concentrations of S. minnesota LPS or (f) E. coli LPS for 3 days before analysis using an MTT assay. Note that 100 μg/ml S. minnesota LPS significantly decreased cell viability. The presented values are mean ± SEM from three different experiments, analysed using ANOVA with Bonferroni correction (, , and was considered significant, CI 95%) to compare between concentrations or an unpaired Student’s t-test (^###, two-tailed, CI 95%) to compare the two chemotypes at a specific concentration.

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