RAW NF-kB/SEAP Reporter Cell Line

Description（描述）

The RAW reporter stable cell line is a stably transfected RAW 264.7 cell line which expresses the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element. RAW 264.7 cells are known to respond to most Toll-like receptor (TLR) ligands, which trigger the NF-kB induction and lead to inflammatory cytokine production. RT-PCR tests also shows that RAW 264.7 cells produce all of the TLR mRNAs except for TLR5 (Figure 1). Using a 96-well plate format assay, the RAW cell line has been validated by TLR ligand stimulation in which all of the TLR ligands except for TLR5 ligand activated the cell line (Figure 2).

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 for this RAW cell line.

Application（应用）

The RAW reporter cell line can be used for screening of TLR agonists or antagonists as well as inhibitory TLR antibody assay.

Product Handling Protocol（产品处理协议）

Note: Please read the entire data sheet before thawing. It is recommended that users follow good tissue culture practice. The RAW cell line is 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 RAW cell line 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 pellet in 10 ml of fresh medium without selection agents.
Note: It is important to grow the RAW cell line at this stage without any selection agents.
7. Transfer the RAW line into a 25-cm2 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 and replace with fresh complete growth medium containing selection agents.
9. At 70-80% confluency, detach the cells by trypsinization and split into new flasks with fresh complete growth medium.
10. Freeze the RAW cell line at 3~4 x 10^6 cells/ml per cryogenic vial. For optimal viability after freezing, freeze cells when they have reached log phase growth (95-98% confluency). Detach by trypsinization at 37oC for 5 min, and harvest by mixing with 3 volumes of fresh medium followed by centrifugation (Step 5). Resuspend the pellet in freeze media (FBS with 10% DMSO). Add 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 the RAW line.
• Wash hands after handling the RAW line and before leaving the lab.
• Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.
• All waste should be considered hazardous.
• Dispose of all liquid waste after each experiment and treat with bleach.

Figure 1. mRNA expression patterns of Toll-like receptors in RAW264.7 cells. Total RNAs were prepared and reverse transcription (RT) was performed to produce cDNAs. PCR was done using the gene-specific primers for mouse TLR1 to TLR9, MD2 as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a house-keeping gene control.

Figure 2. TLR ligand stimulation assay. The RAW cell line was plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with MALP-2 (IMG-2206), Pam3CSK4 (IMG-2201), Poly(I:C) (IMG-2203), LPS (IMG-2204), Flagellin (IMG-2205), R848 (IMG-2208) or mCpG (IMG-2209Mpt) as noted in each graph for 24 h. SEAP was analyzed using IMGENEXs SEAPorter™ Assay Kit (10055K).

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-120	NF-kB/SEAP Stably Transfected RAW Cells	imgenex	1Vial	14044	2-3周