Human TLR9 Reporter HEK293 Cells (NF-κB and IL-8)

NF-κB–SEAP and IL-8–Lucia reporter HEK293 cells expressing human TLR9

Signaling pathways in HEK-Blue-Lucia™ hTLR9 cells

HEK-Blue-Lucia™ hTLR9 cells were engineered from the human embryonic kidney HEK293 cell line to study the Toll-like receptor 9 (TLR9). This important pattern recognition receptor (PRR) recognizes unmethylated CpG dinucleotides, a hallmark of microbial (bacterial, viral, fungal, and parasite) as well as host-derived self DNA [1].

Description

These cells were generated from the HEK-Blue-Lucia™ Null​ cell line harboring two inducible reporter genes. This feature allows the double readout of the NF-κB/AP-1 pathway, by monitoring the SEAP (secreted embryonic alkaline phosphatase) or Lucia luciferase activities. HEK-Blue-Lucia™ hTLR9 cells also stably express the hTLR9 genes. Due to a triple knockout (KO) of TLR3, TLR5, and TNFR, this cell line allows for the independent study of TLR9.

Stimulation of HEK-Blue-Lucia™ hTLR9 cells with TLR9 agonists (e.g. oligonucleotides (ODNs)) triggers the activation of the artificial NF-κB-inducible promoter and the subsequent production of SEAP. It also promotes the expression of Lucia luciferase, which is knocked in (KI) downstream of the endogenous (more physiological) IL-8 promoter (see figures). Of note, their parental cell line HEK-Blue-Lucia™ Null may slightly respond to TLR9-specific agonists due to the weak endogenous expression of TLR9 in HEK293 cells (data not shown).

IL-8 (interleukin 8) is a chemokine produced in response to TLR agonists in an NF-κB/AP-1-dependent manner [1-2]. This feature enables the double readout study of the NF-κB/AP-1 pathway, by monitoring the activity of SEAP and Lucia luciferase using QUANTI-Blue™ Solution (SEAP detection reagent) or QUANTI-Luc™ 4 Lucia/Gaussia (luciferase detection reagent), respectively. Thus, you may choose the readout depending on your laboratory equipment utilizing a spectrophotometer for SEAP or a luminometer for Lucia luciferase detection.
 

Key features

• Stable overexpression of hTLR9
• Verified KO for the TLR3, TLR5, and TNFR genes 
• Functionally validated using a selection of PRR ligands and cytokines
• Readily assessable NF-κB activation by assessing the SEAP and/or Lucia luciferase activities

Applications

• Defining the role of TLR9-dependent NF-κB signaling pathway
• Screening for novel TLR9 agonists and inhibitors
• Choice of readout depending on the laboratory equipment (spectrophotometer for SEAP or luminometer for Lucia luciferase detection).

* Note: This cell line has been renamed. It was formerly known as "HEK-Dual™ hTLR9 (NF/IL8)". The cat. code (hkd-htlr9ni) remains unchanged.

​References:

1. Roebuck KA. 1999. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res:429-38.
2. Ohta K, et al. 2014. Toll-like receptor (TLR) expression and TLR‑mediated interleukin-8 production by human submandibular gland epithelial cells. Mol Med Rep. (5):2377-82.
3. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.

Figures

NF-κB–SEAP response. HEK-Blue-Lucia™ hTLR9 (NF/IL8) and HEK-Blue™ hTLR9 cells were stimulated with various TLR ligands and cytokines: TNF-α (1 ng/ml), Poly(I:C) HMW (TLR3 agonist; 1 µg/ml), FLA‑ST (flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), ODN 2216, ODN 2006, and ODN 2395 (TLR9 agonists; 1 nM). After 24 hour incubation, the NF‑κB‑induced SEAP activity was assessed using QUANTI‑Blue™ Solution and reading the optical density (OD) at 650 nm.

NF-κB (IL-8)–Lucia response using QUANTI-Luc™. HEK-Blue-Lucia™ hTLR9 (NF/IL8) cells were stimulated with 1 nM of the following TLR9 agonists: ODN 2216, ODN 2006, and ODN 2395. After 24 hour incubation, activation of the IL-8 promoter was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Hygromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Quality Control:

• Human TLR9 overexpression has been verified by RT-qPCR and functional assays.
• The triple KO of TLR3, TLR5, and TNFR has been verified by DNA sequencing, PCR, and functional assays.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free. 

Note: HEK-Blue-Lucia™ hTLR9 cells are resistant to Blasticidin, Hygromycin, and Zeocin®. They should be maintained in growth medium supplemented with Hygromycin and Zeocin®.

Contents

• 3-7 x 10⁶ cells in a cryovial or shipping flask.
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Zeocin® (100mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

Toll-Like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [3,4]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [1,3].

TLR9 is mainly expressed in subsets of Dendritic Cells and in B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [3]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [3]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [5].
 

 Get more information about CpG-ODNs Classes.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.

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