|HEK-Dual™ hTLR2 (NF/IL8)||Unit size||Cat. code||Docs||Qty||Price|
Double readout TLR cells
3-7 x 10e6 cells
Human TLR2 (NF-κB-SEAP / KI-[IL-8]Lucia) dual-reporter HEK293 cells
HEK-Dual™ hTLR2 (NF/IL8) cells were generated from HEK-Dual™ Null cells by stable transfection of the human TLR2 (hTLR2) and CD14 genes. Due to the knockout of TLR3 and TLR5, these cells enable the study of hTLR2 signaling without interference from other TLRs. They respond to very low concentrations of TLR2 agonists such as the synthetic lipopeptide Pam3CSK4. 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™ hTLR2 (NF/IL8) and HEK-Blue™ hTLR2 cells were stimulated with various TLR agonists: FSL-1 (TLR2 agonist; 30 pg/ml), Pam3CSK4 (TLR2 agonist; 30 pg/ml), HKLM (TLR2 agonist; 1x107 cells/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™ hTLR2 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™ hTLR2 (NF/IL8) cells were stimulated with TLR2 agonists: FSL-1 (TLR2 agonist; 30 pg/ml), Pam3CSK4 (TLR2 agonist; 30 pg/ml) and HKLM (1x107 cells/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).
EC50 values calculated for the NF-kB response using QUANTI-Blue™. HEK-Dual™ hTLR2 (NF/IL8) and HEK-Blue™ hTLR2 cells were stimulated for 24 hours with various TLR2 agonists: FSL-1, Pam3CSK4 and HKLM.
|Cell line||EC50 for FSL-1 (pg/ml)||EC50 for Pam3CSK4 (pg/ml)||EC50 for HKLM (cells/ml)|
|HEK-Dual™ hTLR2 (NF/IL8)||0.3 ± 0.1||2.5 ± 0.1||0.5 x106 ± 0.1 x106|
|HEK‑Blue™ hTLR2||2.9 ± 0.5||26.4 ± 2.9||1.6 x106 ± 0.5 x106|