|HCT116-Dual™ Cells||Unit size||Cat. code||Docs||Qty||Price|
Human NF-κB-SEAP & IRF-Luc Reporter colorectal carcinoma
3-7 x 10e6 cells
Human HCT116 colorectal carcinoma - NF-κB & IRF reporter cells
HCT116-Dual™ cells are adherent epithelial cells that have been derived from the human HCT116 colorectal carcinoma cell line by stable integration of two inducible reporter constructs.
HCT116-Dual™ cells express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IL-12 p40 minimal promoter fused to five NF-κB and AP-1 binding sites.
HCT116-Dual™ cells also express the Lucia luciferase gene, which encodes a secreted luciferase, under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements.
As a result, HCT116-Dual™ cells allow to simultaneously study the NF-κB pathway, by assessing the activity of SEAP, and the interferon regulatory factor (IRF) pathway, by monitoring the activity of Lucia luciferase.
Stimulation of HCT116-Dual™ cells with the following PAMPs, Tri-DAP (NOD1 ligand, 10 µg/ml), FLA-ST Ultrapure (TLR5, 1 µg/ml), MDP (10 µg/ml), and Poly(I:C) (TLR3 ligand, 1 µg/ml) leads to the activation of NF-κB. IL-1β (100 ng/ml) has been included as positive controls to activate the NF-κB signaling pathway. Non-induced cells (NI), the TLR2 ligand (Pam3CSK4; 300 ng/ml), and the TLR4 ligand (LPS-EB Ultrapure; 104 EU/ml) have been included as negative controls. After a 24h incubation, NF-κB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm.
Stimulation of HCT116-Dual™ cells with the STING agonist 2'3'-cGAMP (30 µg/ml) or RLR ligands, such as transfected poly(I:C) (100 ng/ml) or poly(dA:dT) (100 ng/ml), triggers the IRF pathway. IFN-α (1x104 U/ml) has been included as a positive control to activate the IRF signaling pathway. Non-induced cells (NI), the TLR2 ligand (Pam3CSK4; 300 ng/ml), and the TLR4 ligand (LPS-EB Ultrapure; 104 EU/ml) have been included as negative controls. After a 24h incubation, IRFactivation was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™, a Lucia luciferase detection reagent
- For each lot, proper activation of the NF-κB pathway and IRF pathway is confirmed upon stimulation of HCT116-Dual™ cells by various pathogen associated molecular patterns (PAMPs) known to activate these pathways.
- The stability of this cell line for 20 passages following thawing has been verified.
This product is covered by a Limited Use License (See Terms and Conditions).Back to the top
- 1 vial of HCT116-Dual™ cells (3-7 x 106 cells)
- 1 ml of Blasticidin (10 mg/ml)
- 1 ml of Zeocin™ (100 mg/ml)
- 1 ml of Normocin™ (50 mg/ml)
- 1 pouch of QUANTI-Luc™
- 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
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HCT116-Dual™ cells express numerous pattern recognition receptors (PRRs), including the NOD-like receptors (NLRs) NOD1 and NOD2 [1, 2], the RIG-I-like receptor (RLR) RIG-I [3, 4], and the Toll-like receptors (TLRs) TLR3 and TLR5  but not TLR2 or TLR4 .
Upon recognition of their cognate PAMPs, these receptors induce signaling pathways leading to the activation of the transcription factors NF-kB and/or IRF3/7. Stimulation of HCT116-Dual™ cells with the following PAMPs, Poly(I:C) (TLR3), flagellin (TLR5), Tri-DAP (NOD1), and MDP (NOD2), leads to the activation of NF-kB. Stimulation with RLR ligands, such as transfected poly(I:C) or poly(dA:dT), or the STING agonist, 2’3’-cGAMP, triggers the IRF pathway.
Stimulation with RLR ligands, such as transfected poly(I:C) or poly(dA:dT), or the STING agonist, 2’3’-cGAMP, triggers the IRF pathway.
HCT116-Dual™ cells are resistant to the selectable markers blasticidin and Zeocin™.
1. Rickard DJ. et al., 2014. Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway. PLoS One. 9(5):e96737.
2. Zhao L. et al., 2007. Differential modulation of Nods signaling pathways by fatty acids in human colonic epithelial HCT116 cells. J Biol Chem. 282(16):11618-28.
3 Gack MU. et al., 2008. Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction. PNAS. 105(43):16743-8.
4. Taura M. et al., 2008. p53 regulates Toll-like receptor 3 expression and function in human epithelial cell lines. Mol Cell Biol. 28(21):6557-67.
5. Klimosch SN. et al., 2013. Functional TLR5 genetic variants affect human colorectal cancer survival. Cancer Res. 73(24):7232-42.