HEK-Dual™ hTLR9

Human TLR9 (NF-κB-SEAP / KI-[IL-8]Lucia) dual-reporter HEK293 cells

HEK-Dual™ hTLR9 (NF/IL8) cells were generated from HEK-Dual™ Null cells by stable transfection of the human TLR9 (hTLR9) gene.

Due to the knockout of TLR3 and TLR5, these cells enable the study of hTLR9 signaling without interference from other TLRs.

They respond to very low concentrations of TLR9 agonists, such as the class B CpG oligodeoxynucleotide (CpG ODN) ODN 2006. They do not respond to other TLR agonists or to the cytokine TNF-α (see validation sheet).

HEK-Dual™ hTLR9 (NF/IL8) cells are resistant to hygromycin B, Zeocin™ and blasticidin. They should be maintained in growth medium supplemented with hygromycin B and Zeocin™.

References:

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. 

Figures

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.

Specifications

Antibiotic resistance: blasticidin, hygromycin, Zeocin™.

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin™.

Guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells.
• 100 μl Hygromycin B Gold (100 mg/ml).
• 100 μl Zeocin™ (100mg/ml).
• 1 ml Normocin™ (50 mg/ml).
• 1 pouch of QUANTI-Blue™ (SEAP detection medium).
• 1 pouch of QUANTI-Luc™ (Lucia luciferase detection medium).

Shipped on dry ice