Measuring NF-κB Activation in Inflammatory Cells

HEK-Blue-Null1 cells were seeded in a density of (5 × 10⁴/mL) in 96-well plates and incubated overnight at 37° C. Cells were treated with 1 μM triptolide (Invivogen) (positive control) and different concentrations of nimbolide (1 μM, 0.1 μM, 0.01 μM and 0.001 μM) for 1 h. Then, 100 ng/mL TNF-α (Sigma-Aldrich) were added for 24 h to induce the inflammatory state in the cells. To detect NF-κB activation, 20 μL from the supernatant of cell culture were added to 180 μL of pre-warmed Quanti-Blue detection reagent (Invivogen) per well according to the manufacturer’s instructions. NF-κB activation was detected by measuring SEAP spectrophotometrically at 630 nm using a Tecan reader [64 (link)].