|HEK-Dual™ hTLR5 (NF/IL8)||Unit size||Cat. code||Docs||Qty||Price|
Double readout TLR cells
3-7 x 10e6 cells
Human TLR5 (NF-κB-SEAP / KI-[IL-8]Lucia) dual-reporter HEK293 cells
HEK-Dual™ hTLR5 (NF/IL8) cells were generated from HEK-Dual™ Null cells by stable transfection of the human TLR5 (hTLR5) gene.
Due to the knockout of TLR3, these cells enable the study of hTLR5 signaling without interference from other TLRs. They respond to low concentrations of TLR5 agonist flagellin. They do not respond to other TLR agonists or to the cytokine TNF-α (see validation sheet).
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.
Detection of the NF-kB response using QUANTI-Blue™. HEK-Dual™ hTLR5 (NF/IL8) and HEK-Blue™ hTLR5 cells were stimulated with various TLR agonists: FLA-ST (flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), RecFLA-ST (recombinant flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), FLA-BS (flagellin from B. subtilis, TLR5 agonist; 100 ng/ml), Poly(I:C) HMW (TLR3 agonist; 1 µg/ml) and TNF-α (10 ng/ml). The TLR3 and TNFR activites due to the endogenous expression of these receptors in HEK-Blue™ hTLR5 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™ hTLR5 (NF/IL8) cells were stimulated with TLR5 agonists: FLA-ST (100 ng/ml), FLA-BS (100 ng/ml), and RecFLA-ST (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) fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin™.
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- 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).
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EC50 values calculated for the NF-kB response using QUANTI-Blue™. HEK-Dual™ hTLR5 (NF/IL8) and HEK-Blue™ hTLR5 cells were stimulated for 24 hours with various TLR5 agonists: FLA-ST, recFLA-ST and FLA-BS.
|Cell line||EC50 for FLA-ST (ng/ml)||EC50 for recFLA-ST (ng/ml)||EC50 for FLA-BS (ng/ml)|
|HEK-Dual™ hTLR5 (NF/IL8)||1.4 ± 0.7||200 ± 37.1||568.9 ± 195.5|
|HEK‑Blue™ hTLR5||2.2 ± 1.7||219.4 ± 95.8||410.5 ± 65.6|