STAT3 LeeporterTM Luciferase Reporter-NIH 3T3 Cell Line

Application: Monitor the STAT3 signaling pathway activity. Screen for activators or inhibitors of the STAT3 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 between 80~90% adherent, remove growth medium and passage the cells through trypsinization and centrifugation. At first passage, switch to growth medium containing Puromycin. Note: NIH 3T3 cells should be split before they reach 90% confluence; otherwise, they become self-lifted and aggregate irrevisibly. Precoating the cell plates with 0.1% gelatin may prevent NIH 3T3 cells from self-lifting. During cell trypsinization, cells covered enough with trypsin-EDTA solution should be stayed at 37°C for 10 min without agitation. 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 STAT3 LeeporterTM – NIH 3T3 cells to mIFN-gamma. 1. Plate STAT3 LeeporterTM – NIH 3T3 cells into a white solid-bottom 96-well microplate in 100 μl of growth medium at 1 x 10^5 cells/well and incubate the plate at 37°C in a CO2 incubator for 2-3 hours. 2. Stimulate cells with various concentrations of mIFN-gamma and incubate cells at 37°C in a CO2 incubator for 16 hours. 3. Equilibrate the plate to room temperature for 10 minutes. 4. Add 50 μl of luciferase assay reagent (Abeomics, Cat #17-1101; Refer to the reagent datasheet for the detailed luciferase assay prot°Col) per well. 5. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer. A. Response of STAT3 LeeporterTM – NIH 3T3 cells to IL-6. 1. Plate STAT3 LeeporterTM – NIH 3T3 cells into a white solid-bottom 96-well microplate in 100 μl of growth medium at 1 x 10^5 cells/well and incubate the plate at 37°C in a CO2 incubator for 2-3 hours. 2. Stimulate cells with various concentrations of IL-6 and incubate cells at 37°C in a CO2 incubator for 16 hours. 3. Equilibrate the plate to room temperature for 10 minutes. 4. Add 50 μl of luciferase assay reagent (Abeomics, Cat #17-1101) per well. 5. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.