TNF-alfa LeeporterTM Luciferase Reporter-HEK293 Cell Line

The TNF-alpha LeeporterTM Luciferase Reporter cell line is a stably transfected HEK 293 cell line which expresses Renilla luciferase reporter gene under the transcriptional control of the TNF-alpha promoter. Tumor necrosis factor-alpha (TNF-alpha) is one of the major proinflammatory cytokines and can induce systemic inflammation, apoptotic cell death, sepsis and cachexia. Dysregulation of TNF-alpha induction is often involved in various human diseases including inflammatory bowel disease, cancer and Alzheimer's disease. The TNF-alpha induction by phorbol 12-myristate 13-acetate (PMA) is shown in Figure 1.

Functional Assay

Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.

Dry ice

Immediately upon receipt, store in liquid nitrogen.

Application: Monitor the TNF-alpha induction activity. Screen for activators or inhibitors of the TNF-alpha signaling pathway. Culture conditions: Cells should be grown at 37°C with 5% CO2 using DMEM medium (w/ L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate) supplemented with 10% heat-inactivated FBS and 1% Pen/Strep, plus 3 μg/ml of Puromycin (Note: Puromycin can be omitted during the reporter cell assays) . It is recommended to quickly thaw the frozen cells upon receipt or from liquid nitrogen in a 37°C water-bath, transfer to a tube containing 10 ml of growth medium without Puromycin, spin down cells, resuspend cells in pre-warmed growth medium without Puromycin, transfer resuspended cells to T25 flask and culture in 37°C-CO2 incubator. Leave the T25 flask in the incubator for 1~3 days without disturbing or changing the medium until cells completely recover viability and become adherent. Once cells are over 90% adherent, remove growth medium and passage the cells through trypsinization and centrifugation. At first passage, switch to growth medium containing Puromycin. Cells should be split before they reach complete confluence. To passage the cells, detach cells from culture vessel with Trypsin/EDTA, add complete growth medium and transfer to a tube, spin down cells, resuspend cells and seed appropriate aliquots of cells suspension into new culture vessels. Subcultivation ration = 1:10 to 1:20 weekly. To achieve satisfactory results, cells should not be passaged over 16 times. Functional validation: A. Response of TNF-alpha LeeporterTM – HEK293 cells to phorbol 12-myristate 13-acetate. 1. Harvest TNF-alpha LeeporterTM – HEK293 cells and seed cells into a white solid-bottom 96-well microplate in 100 μl of growth medium at 5 x 10^4 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with different concentrations of phorbol 12-myristate 13-acetate (PMA) . 4. Incubate at 37°C in a CO2 incubator for 16 hours. 5. Equilibrate the plate to room temperature for 10 minutes. 6. Add 50 μl of luciferase assay reagent (Abeomics, Cat #17-1101; Refer to the reagent datasheet for the detailed luciferase assay protocol) per well. 7. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.