TLR8/NF-kB/SEAP Stable Reporter Cell Line

Description（描述）

The TLR8 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor 8 (TLR8) and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element. TLR8 expression in this cell line has been tested by flow cytometry (Fig. 1). Using the 96-well plate format assays, the TLR8 reporter cell line has been validated by DMSO tolerance (Fig.2), cell number titration (Fig. 3), ligand dose response (Fig. 4), and cell stimulation time (Fig. 5).

IMGENEX is pleased to offer the TLR8/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 TLR8 stable cell line are blasticidin and G418.

Application（应用）

The TLR8 reporter line can be used for TLR8-dependent functional assays as well as screening of TLR8 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 TLR8 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 37oC.
2. Thaw the TLR8 reporter 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 reporter cells at this stage without any selection agents.
7. Transfer the TLR8 cell 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 TLR8 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 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 TLR8 reporter line.
• Wash hands after handling the TLR8 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 analysis of TLR8. Intracellular staining of TLR8 in the TLR8 reporter cell line was analyzed by flow cytometry using a PE conjugated TLR8 antibody (IMG-321D). Flow samples were prepared using the Intracellular TLR Staining Flow Kit (10098K). Purple: Cells without antibody; Green: NF-kB/SEAPorter^TM HEK 293 cell line (IML-101) stained with 2 ug anti-TLR8-PE (IMG-321D). Red: TLR8 reporter line stained with anti-TLR8-PE (IMG-321D).

Figure 2. DMSO tolerance evaluation. The TLR8 reporter line was plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with various amounts of R848 (IMG-2208) in the presence of different amounts of DMSO as noted for 24 h. SEAP was then analyzed using IMGENEXs SEAPorter^TM Assay Kit (10055K).

Figure 3. Cell number titration for 96-well plate format assay. The TLR8 reporter line was plated in 96-well plates at different concentrations of cells (100 ul/well) as noted in the figure. After 16 h, cells were stimulated with various amounts of R848, (IMG-2208) for 24 h. Secreted alkaline phosphatase (SEAP) was analyzed using IMGENEXs SEAPorter^TM Assay Kit (10055K). Z factor and Signal vs. Background (S/B) ratio were evaluated for each cell concentration set.

Note: the Z-factor is a measure to quantify the suitability of a particular assay for use in a full-scale, high-throughput screen and is calculated as described previously (1, 2). 

Figure 4. Quantification of the SEAP induced in the R848-stimulated TLR8 reporter cell line. TLR8/NF-kB SEAPorter^TM HEK 293 cells (IML-108) were plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with various amount of R848 (IMG-2208) for 24 h. SEAP was analyzed using the SEAPorter Assay Kit (10055K).
Data Summary: R848 activated the IML-108 cell line in a dose response manner. 

Figure 5. Ligand dose response evaluation. The TLR8 reporter line was plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with various amounts of R848 (IMG-2208) for 24 h. SEAP was analyzed using IMGENEXs SEAPorter^TM Assay Kit (10055K). Dose-responsive percent activation of each sample well was calculated to yield the ligand EC50 value.

Figure 6. Optimal cell stimulation time evaluation. The TLR8 reporter line was plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with various amounts of R848 (IMG-2208) for 6 h, 16 h or 24 h. SEAP was then analyzed using IMGENEXs SEAPorter^TM Assay Kit (10055K).

Reference

1. Zhang, J.H. et al. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67-73.
2. Lee, H.K. et al. (2006). Application of b-lactamase enzyme complementation to the high-throughput screening of Toll-like receptor signaling inhibitors. Mol. Pharmacol.72, 868-875.

订购信息：

货号	名称	产地	规格	报价/元	货期
IML-108	TLR8/NF-kB/SEAP Stable Reporter Cell Line	imgenex	1Vial	18244	2-3周