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

✍ Scribed by Amanda M. Gonzales; Robert A. Orlando

• Publisher: Elsevier
• Year: 2009
• Tongue: English
• Weight: 400 KB
• Volume: 26
• Category: Article
• ISSN: 1871-6784
• DOI: 10.1016/j.nbt.2009.06.982

✦ Synopsis

The transcription factor, Nuclear Factor-kappaB (NF-kappaB), regulates many genes involved in host immunity and cell survival. Unregulated NF-kappaB 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-kappaB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt). To validate this approach, we quantified the activation of NF-kappaB by standard flow cytometry and the HyperCyt platform. Results were comparable with regard to EC(50) values for TNFalpha-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-kappaB activation from a resveratrol-based chemical library. The inhibition of NF-kappaB activation by analog 6q (IC(50) = 19 microm) showed a 3.7-fold improvement over that of resveratrol (IC(50) approximately 70 microm).