TLR3/NF-kB LeeporterTM GFP Reporter-HEK293 Cell Line

Application: Monitor the TLR3 signaling pathway. Screen for activators or inhibitors of the TLR3 signaling pathway. Culture conditions: Cells should be grown at 37oC 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 and 15 μg/ml of 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 37oC 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 37oC-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 TLR3/NF-kB LeeporterTM GFP reporter – HEK293 cells to Poly (I:C) . 1. Harvest TLR3/NF-kB LeeporterTM GFP reporter – HEK293 cells and seed cells into a tissue culture plate (e.g. 96-well plate in 100 ul of growth medium at 5 x 10^4 cells/well, 24-well plate in 500 ul of growth medium at 2.5 x 10^5 cells/well, 12-well plate in 1 ml of growth medium at 5 x 10^5 cells/well, or 6-well plate in 2 ml of growh medium at 1 x 10^6 cells/well. 2. Incubate cells at 37oC in a CO2 incubator for overnight. 3. The next day, stimulate cells with different concentrations of Poly (I:C) (Abeomics, Cat. No.: 15-1012) . 4. Incubate at 37oC in a CO2 incubator for 16 hours. 5.Analyze cells through fluorescence microscopy or flow cytometry.