NF-kB Luciferase Reporter-RAW264.7 Cell Line

Product Name

NF-kB Luciferase Reporter-RAW264.7 Cell Line

SKU/Catalog Number

RC1000

Size

1 Vial, Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.

Description

The NF-kB Luciferase Reporter cell line is a stably transfected RAW 264.7 cell line which expresses Renilla luciferase reporter gene under the transcriptional control of the NF-kB response element. NF-kB is a key transcription factor that is involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis.

Contents

Each vial contains 2 ~ 3 x 10^6 cells in 1 ml of 90% FBS + 10% DMSO.

Storage & Handling

Immediately upon receipt, store in liquid nitrogen. (Ship on dry ice.)

Cite This Product

NF-kB Luciferase Reporter-RAW264.7 Cell Line (Boster Biological Technology, Pleasanton CA, USA, Catalog # RC1000)

Assay Dilutions Recommendation

The recommendations below provide a starting point for assay optimization. Actual working concentration varies and should be decided by the user.

Application:

Monitor the NF-kB signaling pathway.
Screen for activators or inhibitors of the NF-kB 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 (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.

Functional validation: 

A. Response of NF-kB  RAW264.7 cells to  lipopolysaccharide (LPS) 
1. Harvest NF-kB RAW264.7 cells and seed cells into a white solid-bottom 96-well microplate in 100 ul 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.   

Validation Images & Assay Conditions

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