| Assay Method Information | |
| | cell-based NF-kB reporter assay |
| Description: | SH-SY5Y human neuroblastoma cell line was obtained from the American Tissue Culture collection (ATCC). A commercially available expression vector containing the NF-kB promoter enhancer region driving the firefly luciferase gene expression and a second plasmid containing the gene conferring resistance to blasticidin were used in a dual transfection approach to obtain a stable cell line. SH-SY5Y cells stably transfected with pNF-κB-luc/pEF6 were plated at 8,000 per well in a volume of 40 μl DMEM in either Corning white opaque, 384-well plates (Cat. No. 3570, Corning, Inc., Corning, NY) or Corning black clear bottom, 384-well plates (Cat. No. 3712, Corning, Inc., Corning, NY) and treated with test compounds for 24 hr at 37° C. Luciferase activity was measured as a reporter of NF-kB activation using the Bright-Glo Luciferase assay kit (Cat. No. E2620, Promega, Madison, WI) according to the manufacturer's instructions. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability assay kit (Cat. No. G7572, Promega, Madison, WI) according to the manufacturer's instructions. Briefly, cells were equilibrated at room temperature for 30 min prior to the addition of Bright-Glo to the white opaque plates or CellTiter-Glo to the black clear bottom plates. A volume of assay buffer equal to the volume of cell media was added to each well and incubated for five min to allow complete cell lysis. All procedures were performed in the dark. Luminescence was measured by the Synergy4 Multi-detection microplate reader (BioTek, Winooski, VT) within 15 min of lysis. |
| Affinity data for this assay | |
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