4.4. Luciferase Activity Screening

A high-throughput luciferase activity screening method was applied to screen out the putative natural products which regulate promoter luciferase activities. The HEK293 cells were seeded in 24-well plates at the density of 5000 cells/well. When the cells were 80% confluent, they were co-transfected with Renilla vector together with TOPFlash or NF-κB RE reporter vector, respectively. The cells were then treated with chemicals from Selleck small product chemical library at the final concentration of 10 μM. After one-day treatment, the cells were harvested and applied to dual-luciferase assay according to the instructions of dual-luciferase assay reagent (Promega, Madison, WA, USA) with some modifications. In brief, cell lysate was placed into a PerkinElmer VictorTM X2 2030 multilateral reader (PerkinElmer, Waltham, MA, USA) to measure the firefly luciferase activity as well as the renilla luciferase activity. The ratio of firefly luciferase to renilla activity in each sample was revealed as a measurement of the normalized luciferase activity. Triplicated experiments were performed.