Description（描述）

The TLR7 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor7 (TLR7) and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element. The TLR7 reporter cell line has been validated by flow cytometry (Fig. 1) and ligand dose response assay (Fig. 2).

IMGENEX is pleased to offer the TLR7/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 TLR7 stable cell line are blasticidin and G418.

Application（应用）

The TLR7 reporter line can be used for TLR7-dependent functional assays as well as screening of TLR7 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 TLR7 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 TLR7 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 TLR7 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 TLR7 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.

Figure 1. Flow cytometric analysis. Intracellular staining of TLR7 in the TLR7 reporter cell line was analyzed by flow cytometry using PE-conjugated anti-TLR7 antibody (IMG-665D). Flow samples were prepared using the Intracellular TLR Staining Flow Kit (10098K). Purple: Cells without antibody; Green: NF-kB/SEAPorterTM HEK 293 cell line (IML-101) stained with 2 ug anti-TLR7-PE (IMG-665D). Red: TLR7 reporter cell line stained with anti-TLR7-PE (IMG-665D).

Figure 2. Ligand dose response evaluation. The TLR7 reporter cellline 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 SEAPorterTM Assay Kit (10055K). Dose-responsive percent activation of each sample well was calculated to yield the ligand EC50 value.

订购信息：