ISRE Luciferase Reporter-RAW264.7 Cell Line

Reporter Cell Lines

Application: Monitor the JAK/STAT signaling pathway activity.Screen for activators or inhibitors of the JAK/STAT signaling pathway. Culture conditions:Cells should be grown at 37°C with 5% CO2 using DMEM medium supplemented with 10% FBS and 1% Pen/Strep, plus 3 µg/ml of Puromycin.  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 1~2 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.Functional validation:A. Response of ISRE RAW264.7 cells to poly (I:C).1. Harvest ISRE RAW264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 8.5 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 poly (I:C).  4. Incubate at 37°C in a CO2 incubator for 6-16 hours. 5. Add 50 µl of  luciferase assay reagent  per well.  6. Incubate at room temperature for 1-5 minutes and measure luminescence using a microplate luminometer. 

ISRE

The ISRE Luciferase Reporter cell line is a stably transfected RAW264.7 cell line which expresses Renilla luciferase reporter gene under the transcriptional control of the Interferon-Stimulated Response Element (ISRE), so that the cell line is designed to monitor the JAK/STAT signaling pathway activity. This cell line can be activated by type I IFNs as well as certain Toll like receptor ligands capable of induction of IRFs such as TLR3 ligand-poly (I:C). Functional activity of the cell line has been validated by poly (I:C) (Figure 1).