MDA5/NF-kB LeeporterTM Luciferase Reporter-HEK293T Cell Line

Application: Monitor the MDA5 signaling pathway activity. Screen for activators or inhibitors of the MDA5 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 2 μg/ml of Puromycin and 5 μg/ml Blasticidin (Note: Puromycin and Blasticidin 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 and Blasticidin, spin down cells, resuspend cells in pre-warmed growth medium without Puromycin and Blasticidin, 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 and Blasticidin. 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 MDA5 LeeporterTM – HEK293T cells to Poly (I:C) . 1. Harvest MDA5 LeeporterTM – HEK293T 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 various concentrations of the poly (I:C) packed with lipofectamine 2000, which was prepared as follows: i) A 50 μl of 10 mg/ml poly (I:C) (= 500 μg total) is preincubated in 50 μl Opti-MEM (Life Technologies) for 5 min. ii) Similarly, a 20 μl Lipofectamine 2000 (Life Technologies) is preincubated in 80 μl Opti-MEM for 5 min. iii) After 5 min, they are combined together as a total volume of 200 μl and further incubated for 20 min at room temperature. iv) The poly (I:C) packed with Lipofectamine 2000 (A 200 μl total at 2.5 mg/ml) is then used to stimulate cells. 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.