NF-kB/SEAP Stable Reporter Cell Line

Description（产品描述）

The NF-kB/SEAP is a reporter gene assay for the detection of NF-kB activation and contains a cell line modified for this purpose. This cell line is derived from HEK 293 (Human Embryonal Kidney) cells. It is stably transfected with the SEAP (secreted alkaline phosphatase) reporter gene under the transcriptional control of an NF-kB response element. The recombinant cell line is provided frozen.

Placental alkaline phosphatase is one of the most stable isoenzyme, only existing in the placenta of higher primates. These characteristics make placental alkaline phosphatase suitable to use as a reporter gene for the analysis of promoter activity and gene expression in cell culture and animal serum. The natural form of placental alkaline phosphatase (PLAP) is membrane anchored. The recombinant form of placental alkaline phosphatase (secreted alkaline phosphatase, SEAP) is used for reporter gene function. SEAP is created by inserting a translational terminator after amino acid 489 (Berger, et al., Gene 66 (1): 10 (1988). This mutation converts the membrane-bound PLAP protein into the secreted protein.

As a major transcription factor, NF-kB plays a key role in regulating genes responsible for the innate and adaptive immune responses. In unstimulated cells, the NF-kB dimers are held in the cytoplasm by IkBs that masks the nuclear localization signals of NF-kB. Upon cell stimulation, which leads to IkB degradation, NF-kB quickly translocates to the nucleus and activates various genes that have DNA-binding sites for NF-kB.

The NF-kB/SEAP stable HEK 293 cell line is designed to measure NF-kB activation using SEAP protein secreted to the culture media as a read-out with IMGENEX SEAPorter™ Assay kit (10055K). The NF-kB/SEAP stable cells are not only useful in helping with the identification of pro or anti-inflammatory substances, but also can help to assay for proteasome activity since the activation of NF-kB results in the degradation of IkB through the proteasome-dependant pathway.

IMGENEX is pleased to offer the NF-kB Reporter Assay as a service.  

Complete Growth Medium（完全培养基）

DMEM with 4.5 g/L glucose + 10% FBS + 4 mM L-glutamine + 1 mM sodium pyruvate + 100 units/ml penicillin + 100 ug/ml streptomycin + 500 ug/ml G418 (Geneticin)

Note: The selection agent is G418.

Product Handling Protocol（产品处理协议）

Note: Please read the entire data sheet before thawing the cells. It is recommended that users follow good cell culture practice when using the cells. The cells are sterile and all work should be performed under sterile conditions.
1. Prepare a sterile 15-ml tube with 9 ml fresh medium without selection agents pre-warmed at 37oC.
2. Thaw the frozen cell vial quickly in a 37oC water bath, keeping the cap portion out of the water to avoid any possible contamination.
3. Upon thawing, take the vial out of the water and clean it with 70% ethanol to decontaminate.
4. Transfer contents to the 15-ml tube (Step 1) and mix with medium by gentle inversion of tube.
5. Centrifuge at 1,000 RPM for 5 minutes.
6. Remove supernatant and resuspend cells in 10 ml of fresh medium without selection agents.  Note: It is important to grow the cells at this stage without any selection agents.
7. Transfer cells into a 25-cm^2 tissue culture flask and incubate at 37oC in a 95% air-5% CO2 mixture.
8. After cells settle down (in 1-3 days), remove the medium containing minor floating cells and replace with fresh complete growth medium containing selection agents.
9. Whenever the cells are 70-80% confluent, detach the cells by trypsinization and split into new flasks with fresh complete growth medium.
10. Freeze IML cell lines at 3-4x10^6 cells/ml per cryogenic vial. For optimal cell viability after freezing, freeze cells when they have reached log phase growth (95-98% confluency). Detach cells by trypsinization at 37oC for 5 min, and harvest cells by mixing with 3 volumes of fresh medium followed by centrifugation (Step 5). Re-suspend the cell pellet in freeze media (FBS with 10% DMSO). Add cell suspension to cryogenic vials in 1 ml aliquots. Place cryogenic vials, in a tissue culture approved cryogenic vial container, in -80oC freezer for 24-48 hours. After 24-48 hours, move the vials into liquid nitrogen storage.

Safety Considerations（安全注意事项）

Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should be observed.
• Use pipette aids to prevent ingestion and keep aerosols down to a minimum.
• No eating, drinking or smoking while handling cells.
• Wash hands after handling cultures and before leaving the lab.
• Decontaminate work surface with disinfectant or 70% ethanol before and after working with cells.
• All waste should be considered hazardous.
• Dispose of all liquid waste after each experiment and treat with bleach.

Figure 1. Evaluation of the functional activity of the NF-kB/SEAP Stable Reporter Cell Line. NF-kB/SEAPorter^TM HEK 293 cells (IML-101) were plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with 10 ng/ml TNFa, 20 ng/ml Flagellin (IMG-2205) and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 24 h. SEAP was analyzed using the SEAPorter Assay Kit (10055K). Note that HEK 293 cells endogenously express Toll-like receptor 5 (TLR5), responding to flagellin.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-101	NF-kB/SEAP Stable Reporter Cell Line	imgenex	1Vial	9594	2-3周