HEK293 Reporter Cells (NF-κB and IRF)

NF-κB–SEAP and IRF–Lucia reporter HEK293 cells

Signaling pathways in HEK-Dual™ cells

HEK-Dual™ cells are derived from the human embryonic kidney 293 (HEK293) cell line through the stable integration of two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase.

As a result, these cells allow the simultaneous study of the NF-κB pathway, by monitoring the activity of SEAP, and the IRF pathway, by assessing the activity of the secreted Lucia luciferase. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia luciferase detection reagent. 

HEK-Dual™ cells express endogenous levels of various pattern recognition receptors (PRRs), including TLR3, TLR5, NOD1, RIG-I-like receptors (RLR), and STING [in-house data]. They induce the activation of NF-κB in response to cognate TLR/NOD ligands including Poly(I:C),  flagellin, and Tri-DAP. A IRF response is triggered upon stimulation with RLR- and STING-ligands such as 3p-hpRNA and 2’3’-cGAMP, respectively (see figures).

HEK-Dual™ is the control cell line of:

• HEK-Dual™ hTLR3 cells
• HEK-Dual™ hTLR4 cells
• HEK-Dual™ hTLR7 cells
• HEK-Dual™ hTLR8 cells
• HEK-Dual™ hTLR9 cells

Key features

• Functionally validated with a selection of PRR ligands and cytokines
• Readily assessable NF-κB or IRF activation by assessing the SEAP and Lucia luciferase activities

Figures

NF-kB response of HEK-Dual™ cells to various PRR agonists and cytokines. HEK-Dual™ cells were stimulated for 24h with various cytokines and PRR agonists: Human (h)TNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), hIL-1β (NF-κB-positive control, 1 ng/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml) ODN 2006 (TLR9 ligand, 10 µg/ml), Poly(I:C) HMW, complexed with LyoVec™ (LV) (RLR ligand, 1 µg/ml), 3p-hpRNA/LV (RLR ligand, 1 µg/ml), Tri-DAP (NOD1 ligand, 10 µg/ml), 2'3'-cGAMP (STING ligand, 10 µg/ml), and G3-YSD/LV (cGAS ligand, 1 µg/ml). The NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ (1h incubation). Data are shown as optical density (OD) at 630 nm (mean ± SEM).

IRF response of HEK-Dual™ cells to various PRR agonists and cytokines. HEK-Dual™ cells were stimulated for 24h with various cytokines and PRR agonists: Human (h)TNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), hIL-1β (NF-κB-positive control, 1 ng/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml) ODN 2006 (TLR9 ligand, 10 µg/ml), Poly(I:C) HMW, complexed with LyoVec™ (LV) (RLR ligand, 1 µg/ml), 3p-hpRNA/LV (RLR ligand, 1 µg/ml), Tri-DAP (NOD1 ligand, 10 µg/ml), 2'3'-cGAMP (STING ligand, 10 µg/ml), and G3-YSD/LV (cGAS ligand, 1 µg/ml). The IRF response 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, 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:

• The activation of NF-κB and IRF has been verified using functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 ml Zeocin® (100 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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