HEK-Blue™ hTLR2 KO-TLR1/6

NF-κB–SEAP reporter HEK293 cells expressing human TLR2 and knockout for human TLR1 and TLR6

HEK‑Blue™ hTLR2 KO-TLR1/6 cells are a convenient tool to study the TLR2 signaling pathways as the control for HEK‑Blue™ hTLR2-TLR1 and HEK‑Blue™ hTLR2-TLR6 cell lines.

TLR2 plays a pivotal role in detecting a diverse range of pathogen-associated molecular patterns (PAMPs) [1]. 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 [2]. 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 [3].

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.

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 an NF-κB activator, such as IL-1β or TNF-α. hTLR2 KO-TLR1/6 cells do not respond to TLR2 ligands, including heat-killed bacteria, di-, and triacylated lipoproteins. 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. 

To investigate TLR2 signaling pathways, HEK‑Blue™ hTLR2-TLR1 and HEK‑Blue™ hTLR2-TLR6 cell lines are also available.

Read our review on TLR2

References:

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.
75(3):467-77. 
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.

Figures

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 1 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.

Specifications

Antibiotic resistance: blasticidin, hygromycin, Zeocin®

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™

Quality Control:

• HEK-Blue™ hTLR2 KO-TLR1/6 cells have been stimulated by various pathogen recognition receptor (PRR) agonists. As expected, these cells produce SEAP in response to NF-κB activators, such as IL-1β or TNF-α.
• The expression of human TLR2 (hTLR2) and CD14 in this cell line has been validated using fluorescence-activated cell sorting (FACS).
• Biallelic TLR1 and TLR6 knockouts have been verified by functional assays, PCR and DNA sequencing.
• Cell line stability for 20 passages following thawing has been verified.
• These cells are guaranteed mycoplasma-free.

Contents

• 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)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

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