IL-8 Luciferase Reporter-RAW264.7 Cell Line

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 (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 1~2 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. Note: RAW264.7 cells may not be detached well by trypsinization only. So you may need to use a cell scraper to harvest the trypsinized cells.
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 IL-8  RAW264.7 cells to lipopolysaccharide (LPS)
1. Harvest IL-8  RAW264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 µl of growth medium at 8.5 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 LPS.
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.