Application: Monitor the STAT6 signaling pathway activity. Screen for activators or inhibitors of the STAT6 signaling pathway. Culture conditions: Cells should be grown at 37°C with 5% CO2 using RPMI medium supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 10 mM HEPES and 1% Pen/Strep, plus 5 ng/ml mIL-3 (Note: mIL-3 is essential for Ba/F3 cell maintenance), plus 3 μg/ml of Puromycin. (Note: The parental Ba/F3-Puro cell line (Part #14135CCL) is a blank vector-transfected stable cell line which also requires 3 μg/ml of Puromycin for cell culture maintenance! 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. Monitor the cell viability by counting cells daily for 1~3 days until cells completely recover viability as cells are doubling daily. Once cells are over 90% confluent, harvest cells by centrifugation and passage cells. At first passage, switch to growth medium containing Puromycin. Cells should be split before they reach complete confluence. To passage the cells, transfer cells to a tube, spin down cells, resuspend cells and seed appropriate aliquots of cell 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 STAT6 LeeporterTM – Ba/F3 cells to mIL-4. 1. Harvest STAT6 LeeporterTM – Ba/F3 cells and seed cells into a white solid-bottom 96-well microplate in 100 μl of growth medium without IL-3 at 1 x 10^5 cells/well. 2. Right after plating cells, stimulate cells with various concentrations of mouse IL-4 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 protocol) per well. 5. Read the plate in 1-5 minutes to measure luminescence using a microplate luminometer.
