Human TLR9 expressing HEK293 Reporter Cells

HEK-Blue-Lucia™ hTLR9 Unit size Cat. code Docs Qty Price
Human TLR9 expressing double NF-κB–readout HEK293 reporter cells
3-7 x 10e6 cells

Notification: This cell line has been renamed. It was formerly known as "HEK-Dual™ hTLR9 (NF/IL8)". The cat. code (hkd-htlr9ni) remains unchanged.
This product is for internal research use only. Additional rights may be available. Please visit InvivoGen’s Terms and Conditions.

Human TLR9 expressing HEK293 reporter cells

Signaling pathways in HEK-Blue-Lucia™ hTLR9 cells
Signaling pathways in HEK-Blue-Lucia™ hTLR9 cells

InvivoGen offers a human embryonic kidney 293 (HEK293)-derived cell line, specifically designed to assess the distinct role of the human Toll-like receptor 9 (hTLR9):

— HEK-Blue-Lucia™ hTLR9 cells* 

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.

Toll-Like Receptor 9 (TLR9) is one of the most studied pattern recognition receptors (PRRs) for nucleic acid sensing. It is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses [3].

More details More details


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


  • 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).


* formerly named HEK-Dual™ hTLR9 (NF/IL8) cells



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.



NF-kB - SEAP response
NF-kB - SEAP response

NF-κB–SEAP response. HEK-Blue-Lucia™ hTLR9  and HEK-Blue™ hTLR9 cells were stimulated with various TLR agonists: DN2006 (class B CpG ODN, the ligand of choice for hTLR9; 1 μg/ml), ODN1826 (class B CpG ODN, the ligand of choice for murine TLR9; 1 μg/ml), ODN2216 (class A CpG ODN that preferentially binds to hTLR9; 1 μg/ml), Poly(I:C) (TLR3 agonist; 3 μg/ml), FLA-ST (flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), and TNF-α (10 ng/ml). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

NF-κB (IL-8) - Lucia response
NF-κB (IL-8) - Lucia response

NF-κB (IL-8)–Lucia response using QUANTI-Luc™. HEK-Blue-Lucia™ hTLR2 cells were stimulated with TLR9 agonists: ODN2006 (1 μg/ml), ODN1826 (1 μg/ml), and ODN2216 (1 μg/ml). After 24h incubation, activation of the IL-8 was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

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Antibiotic resistance: BlasticidinHygromycinZeocin®

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 BlasticidinHygromycin, and Zeocin®. They should be maintained in growth medium supplemented with Hygromycin and Zeocin®.


These cells are covered by a Limited Use License (See Terms and Conditions).

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

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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.



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.
5Bauer 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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