Double NF-κB–Readout HEK293 Reporter Cells

Double NF-κB–readout HEK293 reporter cells

Signaling pathways in HEK-Blue-Lucia™ Null cells

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

— HEK-Blue-Lucia™ Null cells*

These cells express two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase. Stimulation of HEK-Blue-Lucia™ Null cells with TLR agonists 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. 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.

HEK-Blue-Lucia™ Null cells also feature a triple knockout (KO) of TLR3, TLR5, and TNFR. Therefore, this cell line allows for the independent study of one single TLR when used in combination with:

• HEK-Blue-Lucia™ hTLR2 cells
• HEK-Blue-Lucia™ hTLR3 cells
• HEK-Blue-Lucia™ mTLR4 cells
• HEK-Blue-Lucia™ hTLR5 cells
• HEK-Blue-Lucia™ mTLR7 cells
• HEK-Blue-Lucia™ hTLR9 cells

Key features:

• Verified KO for the TLR3, TLR5, 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

*Note: This cell line has been renamed. It was formerly known as "HEK-Dual™ Null". The cat. code (hkd-nullni) remains unchanged.

​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.

Specifications

Antibiotic resistance: Blasticidin, 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:

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

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

Contents

• 3-7 x 10⁶ cells in a cryovial or shipping flask.
• 1 ml of Zeocin® (100 mg/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)

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