HEK-Blue™ hTLR2-TLR1 Cells
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SEAP reporter 293 cells expressing the human TLR2 and TLR1 genes
3-7 x 10e6 cells
Human TLR2+TLR1 / NF-κB / SEAP reporter HEK293 cells
HEK‑Blue™ hTLR2-TLR1 cells are a convenient tool to study the TLR2/TLR1 signaling pathway and to screen for specific ligands of the TLR2/TLR1 heterodimer.
TLR2 plays a pivotal role in detecting a diverse range of pathogen-associated molecular patterns (PAMPs) . At the cell surface, TLR2 forms a heterodimer with co-receptors TLR1 or TLR6, depending upon either tri- or diacylation of the ligand. Downstream signaling of both TLR2 heterodimers has been shown to be enhanced when in association with CD14 . Once a ligand binds to either TLR2-TLR1 or TLR2-TLR6, a MyD88-dependent activation of NF-κB and AP-1 occurs, ultimately leading to an innate immune response. Importantly, the ability for TLR2 to form heterodimers not only expands the range of PAMPs that it recognizes but can also lead to divergent responses depending on the heterodimer involved .
From HEK293 cells, which endogenously express TLR1 and TLR6, the following engineering work has been performed:
- An NF-κB/AP-1-inducible SEAP reporter gene has been introduced, to allow easy visual monitoring of NF-κB activation.
- Human TLR2 and CD14 genes have been stably transfected.
- Endogenous TLR1 and TLR6 genes have been neutralized by a double knockout.
- Exogenous human TLR1 cDNA has been stably introduced.
Stable expression of the SEAP (secreted embryonic alkaline phosphatase) gene, which is under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites, is triggered by the binding of triacylated lipoproteins, such as Pam3CSK4, to the TLR2-TLR1 heterodimer that activates NF-κB and AP-1. The levels of SEAP can be easily determined with HEK-Blue™ Detection, a cell culture medium that allows real-time detection of SEAP. HEK-Blue™ Detection is a one-step procedure and is applicable to high-throughput screening. SEAP activity can also be assessed using the alkaline phosphatase detection reagent, QUANTI-Blue™. This assay allows the same cell cultures to be repeatedly sampled for kinetic studies or further experimentation.
1. Oliveira-Nascimento L. et al., 2012. The Role of TLR2 in Infection and Immunity. Front Immunol. 3:79.
2. Lotz S. et al., 2004. Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2. J Leukoc Biol.
3. Nguyen MT. et al., 2017. Lipid moieties on lipoproteins of commensal and non-commensal staphylococci induce differential immune responses. Nat Commun. 8(1):2246.
HEK-Blue™ hTLR2, HEK-Blue™ hTLR2‑TLR1, HEK-Blue™ hTLR2‑TLR6 and HEK-Blue™ hTLR2 KO-TLR1/6 cells were incubated in HEK-Blue™ Detection medium and stimulated with 10 ng/ml Pam3CSK4, 0.01 ng/ml Pam2CSK4 and FSL-1, 3 x 106 cells/ml HKLM (heat killed L. monocytogenes; Gram+ bacteria) and HKPA (heat killed P. aeruginosa; Gram- bacteria), and 1 ng/ml of recombinant human IL-1β. After 24h the level of NF-κB-induced SEAP was determined by reading the optical density (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™
- HEK-Blue™ hTLR2-TLR1 cells have been stimulated by various pathogen recognition receptor (PRR) agonists. As expected, TLR2-TLR1 agonists induced the production of SEAP.
- The expression of human TLR2 (hTLR2) and CD14 in this cell line has been validated using fluorescence-activated cell sorting (FACS).
- The expression of hTLR1 has been confirmed by qRT-PCR.
- Cell line stability for 20 passages following thawing has been verified.
- These cells are guaranteed mycoplasma-free.