|A549-Dual™ Cells||Unit size||Cat. code||Docs||Qty||Price|
Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma
3-7 x 10e6 cells
Human NF-κB-SEAP & IRF-Luc Reporter lung carcinoma
A549-Dual™ cells are adherent epithelial cells that have been derived from the human A549 lung carcinoma cell line by stable integration of two inducible reporter constructs. The A549 cell line is a well-characterized cellular model for asthma, allergies and respiratory infections.
A549-Dual™ cells express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB binding sites.
A549-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, A549-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 A549-Dual™ cells with the following PAMPs, Pam3CSK4 (TLR2 ligand, 300 ng/ml) Poly(I:C) (TLR3 ligand, 3 µg/ml ), FLA-ST Ultrapure (TLR5, 300 ng/ml), leads to the activation of NF-κB. TNF-α (1 ng/ml) and IL-1β (1 ng/ml) have been included as positive controls to activate the NF-κB signaling pathway. Non-induced cells (NI), the TLR4 ligand (LPS-EB Ultrapure; 103 EU/ml), and the TLR7/8 ligand (R848; 10 µg/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 A549-Dual™ cells with RLR ligands, such as transfected poly(I:C) (100 ng/ml), 5'ppp-dsRNA (1 µg/ml) or poly(dA:dT) (100 ng/ml) triggers the IRF pathway. The STING agonist 2'3'-cGAMP (30 µg/ml) leads to low-level IRF induction in A549-Dual™ cells. IFN-α (1x104 U/ml) has been included as a positive control to activate the IRF signaling pathway. Non-induced cells (NI), the TLR4 ligand (LPS-EB Ultrapure; 103 EU/ml), and the TLR7/8 ligand (R848; 10 µg/ml) have been included as negative controls. After a 24h incubation, IRF activation was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™, a Lucia luciferase detection reagent.
- The stability of this cell line for 20 passages following thawing has been verified.
- For each lot, proper activation of the NF-κB pathway and IRF pathway is confirmed upon stimulation of A549-Dual™ cells by various pathogen associated molecular patterns (PAMPs) known to activate these pathways.
These products are covered by a Limited Use License (See Terms and Conditions).Back to the top
- 1 vial of A549-Dual™ cells (3-7 x 106 cells) in Freezing Medium
- 1 ml of Zeocin® (100 mg/ml)
- 1 ml of Blasticidin (10 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 pouch of QUANTI-Luc™
Shipped on dry ice (Europe, USA, Canada and some areas in Asia)Back to the top
A549-Dual™ cells express numerous pattern recognition receptors (PRRs), including the RIG-I-like receptor (RLR) RIG-I [1, 2], and the Toll-like receptors (TLRs) TLR2 , TLR3 [4, 5] and TLR5  but not 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 A549-Dual™ cells with the following PAMPs, Pam3CSK4 (TLR2) Poly(I:C) (TLR3), flagellin (TLR5), leads to the activation of NF-kB. IL-1b or TNF-a can be used as positive controls to activate the NF-kB signaling 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. IFN-a can be used as positive controls to activate the IRF signaling pathway.
A549-Dual™ cells are resistant to the selectable markers blasticidin and Zeocin™.
1. Kolokoltsova OA. et al., 2014. RIG-I enhanced interferon independent apoptosis upon Junin virus infection. PLoS One. 9(6):e99610.
2. Hagmann CA. et al., 2013. RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells. PLoS One. 8(4):e62872.
3 Slevogt H. et al., 2007. Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response. Cell Microbiol. 9(3):694-707.
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. Tissari J. et al., 2015. IFN-alpha enhances TLR3-mediated antiviral cytokine expression in human endothelial and epithelial cells by up-regulating TLR3 expression J Immunol. 174(7):4289-94.
6. 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. 4:33.