Human TLR9 Dual-Reporter HEK293 Cells
|HEK-Dual™ hTLR9 (NF/IL8)||Unit size||Cat. code||Docs||Qty||Price|
Double readout TLR cells
3-7 x 10e6 cells
Human TLR9 (NF-κB-SEAP / KI-[IL-8]Lucia) dual-reporter HEK293 cells
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InvivoGen offers a human embryonic kidney 293 (HEK293)-derived cell line, specifically designed for the study of human TLR9 (Toll-Like Receptor 9)-induced NF-κB signaling pathway:
• HEK-Dual™ hTLR9 cells
HEK-Dual™ hTLR9 cells overexpress the human TLR9 (hTLR9) gene. Like their parental cells, HEK-Dual™ Null, they are knockout of TLR3, TLR5, and TNFR genes (all of which are endogenously expressed in HEK293 cells). They also feature two inducible reporter systems:
— a SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of an NF-κB inducible promoter
— a secreted Lucia luciferase reporter gene placed under the control of the endogenous IL-8 promoter; the coding sequence of IL-8 was replaced by the Lucia luciferase ORF using knock-in technology. IL-8 is a chemokine produced in response to TLR agonists in an NF-κB and AP-1 dependent-manner [1, 2].
The two reporter proteins, SEAP and Lucia luciferase can be readily measured in the supernatant by using QUANTI-Blue™ Solution and QUANTI‑Luc™, respectively.
Unlike their parental cells which weakly express TLR9, HEK-Dual™ hTLR9 cells are responsive to stimulation with TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs).
Toll-Like Receptor 9 (TLR9) is one of the most studied pattern recognition receptors (PRRs) for nucleic acids. It 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 [3-5]. TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs) [3,5].
- Verified overexpression of TLR9 gene by RT-PCR
- Functionally validated with a selection of TLR9 agonists
- Readily assessable SEAP and Lucia reporter activity
- Study of the NF-κB-dependent TLR9 signaling pathway
- Screening of novel specific activators or inhibitors of the TLR9 signaling pathway
1. Ohta K. et al., 2014. TLR-mediated interleukin-8 production by human submandibular gland epithelial cells. Mol Med Rep. 10(5):2377-82.
2. Roebuck KA. et al., 1999. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res. 19(5):429-38.
3. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
4. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
5. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
Detection of the NF-kB response using QUANTI-Blue™. HEK-Dual™ hTLR9 (NF/IL8) and HEK-Blue™ hTLR9 cells were stimulated with various TLR agonists: ODN2006 (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). The TLR3, TLR5 and TNFR activities due to the endogenous expression of these receptors in HEK-Blue™ hTLR9 cells are shown in gray. After 24 hour incubation, NF‑kB‑induced SEAP activity was assessed using QUANTI‑Blue™ and reading the optical density (OD) at 655 nm.
Detection of the IL-8 response using QUANTI-Luc™. HEK-Dual™ hTLR9 (NF/IL8) cells were stimulated with TLR9 agonists: ODN2006 (1 μg/ml), ODN1826 (1 μg/ml) and ODN2216 (1 μg/ml). 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™, a Lucia luciferase detection reagent. The activation of the IL-8 promoter is expressed as fold increase relative to untreated cells which was calculated by dividing the RLUs for the treated cells by the RLUs for the untreated cells.
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™
- Human TLR9 expression has been verified by RT-PCR and functional assays.
- The biallelic replacement of the human interleukin-8 (IL-8) open reading frame (ORF) with the Lucia luciferase reporter ORF was verified by PCR and sequencing.
- The inability to produce IL-8 has been confirmed by ELISA.
- TLR3, TLR5, and TNFR triple knockout has been verified by DNA sequencing and functional assays.
- The stability for 20 passages, following thawing, has been verified.
- These cells are guaranteed mycoplasma-free.
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- 3-7 x 106 HEK-Dual™ hTLR9 cells in a cryovial or shipping flask
- 1 ml of Hygromycin B Gold (100 mg/ml)
- 1 ml of Zeocin™ (100 mg/ml)
- 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
- 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 pouch of QUANTI-Luc™ (Lucia luciferase detection medium)
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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 . The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) . They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively .
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.
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.