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 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 STAT3 LeeporterTM – HEK293 cells to IL-6 (Figure 1) . 1. Harvest STAT3 LeeporterTM – HEK293 cells and seed cells into a white solid-bottom 96-well microplate in 100 μl of growth medium at 5 x 104 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with different concentrations of IL-6. 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. B. Inhibition of IL-6-induced STAT3 activity (Figure 2) . 1. Harvest STAT3 LeeporterTM – HEK293 cells and seed cells into a white solid-bottom 96-well microplate in 100 ul of growth medium at 5 x 104 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for overnight. 3. The next day, pre-treat cells with different concentrations of anti-IL-6Ra antibody (MAB227, R & D Systems) for 1 hour. 4. Add 10 ng/ml IL-6 and incubate cells 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) per well. 7. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer. C. Response of STAT3 LeeporterTM – HEK293 cells to Oncostatin-M (Figure 3) . 1. Harvest STAT3 LeeporterTM – HEK293 cells and seed cells into a white solid-bottom 96-well microplate in 100 μl of growth medium at 5 x 104 cells/well. 2. Incubate cells at 37°C in a CO2 incubator for overnight. 3. The next day, stimulate cells with different concentrations of Oncostatin-M. 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) per well. 7. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.
