Human TLR2 Reporter HEK293 Cells (NF-κB)
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Human TLR2 expressing HEK293 reporter cells (NF-κB pathway)
3-7 x 10e6 cells
NF-κB–SEAP reporter HEK293 cells expressing human TLR2
HEK-Blue™-hTLR2 cells were obtained by co-transfection of the human TLR2 and SEAP (secreted embryonic alkaline phosphatase) genes into HEK293 cells.
The SEAP reporter gene is placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites.
Additionally, the CD14 co-receptor gene was transfected into these cells to enhance the TLR2 response.
Stimulation with a TLR2 ligand activates NF-κB and AP-1 which induce the production of SEAP.
Levels of SEAP can be easily determined with HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP.
The parental cell line of HEK-Blue™ hTLR2 cells is HEK-Blue™ Null1 cells.
HEK-Blue™ hTLR2 cells were stimulated with various TLR and NOD agonists: Pam3CSK4 (100 ng/ml), Poly(I:C) (50 ng/ml), LPS-EB ultrapure (100 ng/ml), recombinant flagellin from S. typhimurium (10 ng/ml), CL264 (1 µg/ml), CL097 (3 μg/ml), ssRNA40/LyoVec™ (5 μg/ml), ODN 2006 (10 μg/ml), C12-iE-DAP (100 ng/ml), L18-MDP (100 ng/ml), and TNF-α (100 ng/ml). After 18h incubation (24h incubation for CL264, C12-iE-DAP and L18-MDP ligands), NF-κB-induced SEAP activity was assessed using QUANTI-Blue™ and by reading the OD at 655 nm.
HEK-Blue™ hTLR2 and HEK-Blue™ Null1 (control) cells were incubated in HEK-Blue™ Detection medium and stimulated with 0.1 ng/ml FSL-1 (TLR2/6), 10 ng/ml Pam3CSK4 (TLR1/2), 107 cells/ml HKLM, 1 µg/ml LTA-SA, 10 ng/ml LPS-PG Ultrapure or 1 µg/ml PGN-BS. After 24h incubation, the levels of NF-κB-induced SEAP were determined by reading the OD at 655 nm.
HEK-Blue™ TLR2 cells were pre-incubated for 1h with increasing concentrations of anti-hTLR2-IgA, anti-hTLR6-IgG or anti-hTLR1-IgG then stimulated with 0.5 ng/ml of Pam3CSK4 for 18h. NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ by reading the OD at 655 nm.
HEK-Blue™ TLR2 cells were pre-incubated for 1h with increasing concentrations of anti-hTLR2-IgA, anti-hTLR6-IgG or anti-hTLR1-IgG then stimulated with 0.1 ng/ml of FSL-1 for 18h. NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ by reading the OD at 655 nm.
Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™
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- 1 vial containing 3-7 x 106 cells
- 2 x 1 ml 250X HEK-Blue™ Selection
- 1 ml Normocin™ (50 mg/ml)
- 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)
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