HEK-Blue-Lucia™ TNF-α Cells

Human TNF-α detecting double NF-κB–readout HEK293 reporter cells

Signaling pathways in HEK-Blue-Lucia™ TNF-α cells

InvivoGen offers a human embryonic kidney 293 (HEK293)-derived cell line, specifically designed to detect the bioactive human tumor necrosis factor-alpha (TNF-α):

— HEK-Blue-Lucia™ TNF-α cells* 

These cells were generated from the HEK293 cell line through the stable integration of two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) or Lucia luciferase. This feature allows the double readout 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. 
 

Stimulation of HEK-Blue-Lucia™ TNF-α cells with recombinant human (h)TNF-α triggers the activation of the NF-κB-inducible promoter and the production of SEAP as well as Lucia luciferase. 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™  TNF-α cells also feature a knockout (KO) of the adaptor protein MyD88. Therefore, this cell line allows for the isolated study of the TNF-α-dependent NF-κB response without the interference of other receptors (e.g. TLR5, IL1R). As expected, these cells do not respond to flagellin (TLR5 ligand) or IL-1β (IL1R ligand). 

TNF-α is a multi-functional pro-inflammatory cytokine involved in the regulation of a wide spectrum of biological processes, such as cell proliferation, differentiation, and apoptosis [1].

 More details

Key features:

• Fully functional TNF-α signaling pathway
• Verified KO of the MyD88 gene
• Do not respond to IL-1β
• Functionally validated using a selection of PRR ligands and cytokines
• Readily assessable SEAP and Lucia luciferase reporter activity

Applications:

• Detecting human TNF-α
• Screening of novel anti-hTNF-α antibodies 
• Choice of readout depending on the laboratory equipment (spectrophotometer for SEAP or luminometer for Lucia luciferase detection).

* formerly named HEK-Dual™ TNF-α cells

1. Steeland S, Libert C, Vandenbroucke RE. 2018. A New Venue of TNF Targeting. Int J Mol Sci.;19(5):1442. 

Figures

Dose-response of HEK-Blue-Lucia™ TNF-α cells to recombinant human TNF-α. Cells were stimulated with increasing concentrations of recombinant human (h)TNF-α. After overnight incubation, (A) the NF-κB-induced SEAP activity was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are shown as optical density (OD) at 630 nm (mean ± SEM). (B) The activation of the IL-8 promoter was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent. The activation of the IL-8 promoter is expressed as fold increase relative to untreated cells (mean ± SEM).

Response profile of HEK-Blue-Lucia™ TNF-α cells. Cells were incubated for 24 hours with cytokines and various TLR agonists: hTNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), hIL-1β (1 µg/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 1 µg/ml), R848 (TLR7/8 ligand, 10 µg/ml), and ODN 2006 (TLR9 ligand, 1 µg/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed using QUANTIBlue™. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

Response profile of HEK-Blue-Lucia™ TNF-α cells (Lucia). Cells were incubated for 24 hours with cytokines and various TLR agonists: hTNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), hIL-1β (1 µg/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 1 µg/ml), R848 (TLR7/8 ligand, 10 µg/ml), and ODN 2006 (TLR9 ligand, 1 µg/ml).After 24h incubation, the activation of the IL-8 promoter 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: 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:

• Reporter gene activities in response to TNF-α and various cytokines have been validated using functional assays.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free. 

Detection range for hTNF-α when using:

• QUANTI-Blue™ Solution: 1 ng - 1 µg/ml
• QUANTI-Luc™ 4 Gaussia/Lucia: 0.5 ng - 1 µg/ml

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 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)

Details

Tumor necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine produced by several types of cells, predominantly activated macrophages [1.] TNF-α plays an important role in the immune response to microbial invasions and in the necrosis of specific tumors. Of note,  as a potent mediator of inflammation, TNF-α has been implicated in the pathogenesis of several autoimmune and inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease [1,2].

TNF-α exists in two forms; a type II transmembrane protein and a mature soluble protein. The TNF-α transmembrane protein is proteolytically cleaved to yield a soluble protein [3], which subsequently forms a non-covalently linked homotrimer in solution. TNF-α binds two receptors TNFR1 and TNFR2 inducing signaling that involves TRADD, TRAF2, and RIP, and leads to the activation of the NF-κB and the MAPK pathways [4]. 

Interleukin 1 beta (IL-1β) is another inflammatory cytokine that triggers these pathways following the binding to its receptor IL-1RI and the recruitment of MyD88. Both TNF-α and IL-1β receptors are expressed in HEK293 cells. HEK-Dual™ TNF-α Cells are rendered unresponsive to IL-1β by stable knock-out of the MyD88 gene.

1. Sedger L. & McDermott M., 2014. TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants - past, present and future. Cytokine Growth Factor Rev. 25(4):453-72.
2. Li P. et al., 2017. Drugs for Autoimmune Inflammatory Diseases: From Small Molecule Compounds to Anti-TNF Biologics.Front Pharmacol .8:460.
3. Kriegler M. et al., 1988. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell. 53(1):45-53.
4. Wajant H. et al., 2003. Tumor necrosis factor signaling. Cell Death Differ. 2003 10(1):45-65.

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