|HEK-Dual™ mTLR4 (NF/IL8)||Unit size||Cat. code||Docs||Qty||Price|
Double readout TLR cells
3-7 x 10e6 cells
HEK-Dual™ mTLR4 (NF/IL8) cells were generated from HEK-Dual™ Null cells by stable transfection of the mouse TLR4 (mTLR4), MD2 and CD14 genes.
Due to the knockout of TLR3 and TLR5, these cells enable the study of mTLR4 signaling without interference from other TLRs.
They respond to very low concentrations of TLR4 agonists such as lipopolysaccharide (LPS). 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™ mTLR4 (NF/IL8) and HEK-Blue™ mTLR4 cells were stimulated with various TLR agonists: LPS-EB Ultrapure (LPS from E. coli 0111:B4, TLR4 agonist; 1 ng/ml), LPS-EK Ultrapure (LPS from E. coli K12, TLR4 agonist; 1 ng/ml), MPLA Synthetic (TLR4 agonist; 1 ng/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™ mTLR4 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™ mTLR4 (NF/IL8) cells were stimulated with TLR4 agonists: LPS-EB Ultrapure (1 ng/ml), LPS-EK Ultrapure (1 ng/ml) and MPLA Synthetic (1 ng/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™ mTLR4 (NF/IL8) and HEK-Blue™ mTLR4 cells were stimulated for 24 hours with various TLR4 agonists: LPS-EB UP, LPS-EK UP and MPLAs.
|Cell line||EC50 for LPS-EB UP (ng/ml)||EC50 for LPS-EK UP (ng/ml)||EC50 for MPLAs (ng/ml)|
|HEK-Dual™ mTLR4 (NF/IL8)||0.06 ± 0.01||1.38 ± 0.35||0.12 ± 0.02|
|HEK‑Blue™ mTLR4||0.06 ± 0.01||1.21 ± 0.36||0.09 ± 0.02|