TLR4/NF-kB/SEAP Stable Reporter Cell Line

Description（描述）

The TLR4 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor 4 (TLR4) and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element. The TLR4 reporter cell line has been validated by flow cytometry (Fig. 1). Functional activity of this cell line has been validated by transfecting the MD2 plasmid and analyzing SEAP activity with IMGENEX�s SEAPorter� Assay Kit (10055K) (Fig. 2).

IMGENEX is pleased to offer the TLR4/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 + 10 ug/ml blasticidin + 500 ug/ml G418 (Geneticin)
Note: The selection agents for the TLR4 stable cell line are blasticidin and G418.

Application（应用）

The TLR4 reporter line can be used for TLR4-dependent functional assays as well as screening of TLR4 agonists or antagonists.

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 TLR4 reporter 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 37^oC.
2. Thaw the TLR4 reporter cell line vial quickly in a 37^oC 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 reporter cells at this stage without any selection agents.
7. Transfer the TLR4 cell line into a 25-cm2 tissue culture flask and incubate at 37^oC 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 TLR4 reporter 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 37^oC 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 -80^oC 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 TLR4 reporter line.
• Wash hands after handling the TLR4 reporter line 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. Flow cytometric analysis. Surface staining of the TLR4 reporter cell line was analyzed by flow cytometry using a PE-conjugated anti-TLR4 antibody (IMG-417D). Flow samples were prepared using the TLR Staining Flow Kit (10099K). Purple: Cells alone; Green: NF-kB/SEAPorter^TM HEK 293 cell line (IML-101) stained with 1 ug anti-TLR4-PE (IMG-417D); Red: TLR4 reporter cell line stained with 1 ug anti-TLR4-PE (IMG-417D).

Figure 2. Evaluation of the functional activity of the TLR4/NF-kB SEAPorter^TM HEK 293 cell line. TLR4/NF-kB SEAPorter^TM HEK 293 cells (IML-104) were plated in 24-well plates at 2 x 10^5 cells/well. After 16 h, cells were transiently transfected with control vector, MD2 plasmid or MD2/CD14 plasmid vector for 24 h. Cells were then stimulated with 0.5 ug/ml LPS (IMG-2204) for 24 h. SEAP was analyzed using the SEAPorter Assay Kit (10055K).
Data Summary: Both MD2 and CD14 are required for the complete LPS-mediated TLR4 activation.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-104	TLR4/NF-kB/SEAP Stable Reporter Cell Line	imgenex	1Vial	18244	2-3周