Human TLR5 HEK293 Reporter Cells (NF-κB and IL-8)

NF-κB–SEAP and IL-8–Lucia reporter HEK293 cells expressing human TLR5

Signaling pathways in HEK-Blue-Lucia™ hTLR5 cells

InvivoGen offers a human embryonic kidney 293 (HEK293)-derived cell line, specifically designed to assess the distinct role of the human Toll-like receptor 5 (hTLR5):

— HEK-Blue-Lucia™ hTLR5 cells* 

These cells were generated from the HEK-Blue-Lucia™ Null​ cell line harboring two inducible reporter genes. This feature allows the double readout of the NF-κB/AP-1 pathway, by monitoring the SEAP (secreted embryonic alkaline phosphatase) or Lucia luciferase activities. HEK-Blue-Lucia™ hTLR5 cells also stably express the hTLR5 gene. Due to a double knockout (KO) of TLR3 and TNFR, this cell line allows for the independent study of TLR5.

Stimulation of HEK-Blue-Lucia™ hTLR5 cells with TLR5 agonists (e.g. flagellin) triggers the activation of the artificial NF-κB-inducible promoter and the subsequent production of SEAP. It also promotes the expression of Lucia luciferase, which is knocked in (KI) downstream of the endogenous (more physiological) IL-8 promoter (see figures).

IL-8 (interleukin 8) is a chemokine produced in response to TLR agonists in an NF-κB/AP-1-dependent manner [1-2]. This feature enables the double readout study of the NF-κB/AP-1 pathway, by monitoring the activity of SEAP and Lucia luciferase using QUANTI-Blue™ Solution (SEAP detection reagent) or QUANTI-Luc™ 4 Lucia/Gaussia (luciferase detection reagent), respectively. Thus, you may choose the readout depending on your laboratory equipment utilizing a spectrophotometer for SEAP or a luminometer for Lucia luciferase detection.
 

Upon recognition of the flagellin of Gram-negative or -positive bacteria, TLR5 mediates the transcriptional induction of proinflammatory cytokines, such as TNF-α [3]. 

 More details

Key features:

• Stable overexpression of hTLR5
• Verified KO for the TLR3 and TNFR genes 
• Functionally validated using a selection of PRR ligands and cytokines
• Readily assessable NF-κB activation by assessing the SEAP and/or Lucia luciferase activities

Applications:

• Defining the role of TLR5-dependent NF-κB signaling pathway
• Screening for novel TLR5 agonists and inhibitors
• Choice of readout depending on the laboratory equipment (spectrophotometer for SEAP or luminometer for Lucia luciferase detection).

* formerly named HEK-Dual™ hTLR5 (NF/IL8) cells

​References:

1. Roebuck KA. 1999. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res:429-38.
2. Ohta K, et al. 2014. Toll-like receptor (TLR) expression and TLR‑mediated interleukin-8 production by human submandibular gland epithelial cells. Mol Med Rep. (5):2377-82.
3. Yoon SI et al. 2012. Structural basis of TLR5-flagellin recognition and signaling. Science. ;335(6070):859-64.

Figures

NF-κB–SEAP response using QUANTI-Blue™ Solution. HEK-Blue-Lucia™ hTLR5  and HEK-Blue™ hTLR5 cells were stimulated with various TLR agonists: FLA-ST (flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), RecFLA-ST (recombinant flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), FLA-BS (flagellin from B. subtilis, TLR5 agonist; 100 ng/ml), Poly(I:C) HMW (TLR3 agonist; 1 µg/ml) and TNF-α (10 ng/ml). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

NF-κB (IL-8)–Lucia response using QUANTI-Luc™. HEK-Blue-Lucia™ hTLR5 cells were stimulated with TLR5 agonists: FLA-ST (100 ng/ml), FLA-BS (100 ng/ml), and RecFLA-ST (1 µg/ml). After 24h incubation, activation of the IL-8 was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Hygromycin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Quality Control:

• Human TLR5 overexpression has been verified by RT-qPCR and functional assays.
• The double KO of TLR3 and TNFR has been verified by DNA sequencing, PCR, and functional assays.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free. 

Note: HEK-Blue-Lucia™ hTLR5 cells are resistant to Blasticidin, Hygromycin, and Zeocin®. They should be maintained in growth medium supplemented with Hygromycin and Zeocin®.

These cells are covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ cells in a cryovial or shipping flask.
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Zeocin® (100mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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

Details

The Toll-like receptor 5 (TLR5) is an important pattern recognition receptor (PRR) of the innate immune system playing an essential role in the respiratory tract, gastrointestinal tract, and liver. It recognizes a wide variety of pathogen-associated molecular patterns (PAMPs), specifically flagellin, the major structural protein of Gram-positive and Gram-negative bacterial flagella [1]. TLR5 is expressed constitutively in epithelial cells and immune cells, such as monocytes and immature dendritic cells (DCs). It is preferentially found on the apical side of respiratory epithelia in both mice and humans [1]. Activation of the receptor stimulates the production of proinflammatory cytokines, such as TNF-α, through signaling via the adaptor protein MyD88 [1-3]. TLR5 can generate a pro-inflammatory signal as a homodimer suggesting that it might be the only TLR participating in flagellin recognition [3]. However, TLR5 may require the presence of a co-receptor or adaptor molecule for efficient ligand recognition and/or signaling [4].

References

1. Yang J. & Yan H. 2017. TLR5: beyond the recognition of flagellin.Cell Mol Immunol. 14(12):1017-1019.
2. Gewirtz AT. et al., 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol, 167(4):1882-5.
3. Hayashi F. et al., 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature, 410(6832):1099-103.
4. Tallant T. et al., 2004. Flagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-kappa B and proinflammatory gene program activation in intestinal epithelial cells. BMC Microbiol. 4(1):33.

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