HEK-Dual™ hTLR9

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.