A Jurkat transcriptional reporter cell line for high-throughput analysis of the Nuclear Factor-κB signaling pathway

The transcription factor, Nuclear Factor-κB (NF-κB), regulates many genes involved in host immunity and cell survival. Unregulated NF-κB activity has been linked to many chronic inflammatory diseases and is an important target for the identification of inhibitors to better manage these disorders. We present a novel screening system to identify NF-κB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt®). To validate this approach, we quantified the activation of NF-κB by standard flow cytometry and the HyperCyt® platform. Results were comparable with regard to EC50 values for TNFα-mediated activation; however, the HyperCyt® platform provided more sensitive signal detection and a greater linear range for detection. To demonstrate the usefulness of this screening tool, we identified a novel inhibitor of NF-κB activation from a resveratrol-based chemical library. The inhibition of NF-κB activation by analog 6q (IC50 = 19 μm) showed a 3.7-fold improvement over that of resveratrol (IC50 ∼ 70 μm).