J774-Dual™ Cells

NF-kB-SEAP & IRF-Luc Reporter Macrophages

J774-Dual™ cells have been derived from the mouse J774.1 macrophage-like cell line by stable integration of two inducible reporter constructs.

J774-Dual™ cells express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site.

J774-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, J774-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.
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 and Gaussia luciferase detection reagent.

Figures

J774-Dual™ cells were stimulated with various PRR agonists known to activate the NF-κB pathway: Pam3CSK4 (TLR1/2 ligand, 1 µg/ml), ultrapure lipopolysaccharide from Escherichia coli K12 (LPS-EK UP, TLR4 ligand, 100 ng/ml), poly(I:C) (TLR3 ligand, 10 ng/ml), ODN1826 (TLR9 ligand, 10 µg/ml) and L18-MDP (NOD2 ligand, 10 µg/ml) NF-κB-induced SEAP activity was assessed using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm.

J774-Dual™ cells were stimulated with various PRR agonists known to activate the IRF pathway: 2'3'-cGAMP (STING ligand, 2 µg/ml), poly(I:C) complexed with the transfection reagent LyoVec™ (LV) (RIG-I/MDA-5 ligand, 30 ng/ml), and 5'ppp-dsRNA (5P-RNA) complexed with LyoVec™ (RIG-I ligand, 1 µg/ml). After 24h, IRF-induced Lucia luciferase activity was determined by measuring relative light units (RLUs) using the QUANTI-Luc™ assay and a luminometer.

Specifications

Antibiotic resistance: Blasticidin and Zeocin®

Guaranteed mycoplasma-free

Quality Control:

• For each lot, proper activation of the NF-κB pathway and IRF pathway is confirmed upon stimulation of J774-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.

Contents

• 1 vial of J774-Dual™ cells (3-7 x 10⁶ cells)
• 1ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of& Normocin™ (50 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 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)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

J774.1 cells express a variety of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) [1, 2],  C-type lectin receptors (CLRs) [2,3], RIG-I-like receptors (RLRs) [4].

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 J774-Dual™ cells with the following PAMPs, Pam3CSK4 (TLR1/2), lipopolisaccharide (TLR4), CpG ODNs (TLR9), L18-MDP (NOD2) and TDB (Mincle), leads to the activation of NF-kB.
Stimulation with RLR ligands, such as transfected poly(I:C) or 5’ppp-dsRNA, or the STING agonist, 2’3’-cGAMP, triggers the IRF pathway.

J774-Dual™ cells are resistant to the selectable markers blasticidin and Zeocin®.

1. Jin M. et al., 2011. Effects of chondroitin sulfate and its oligosaccharides on toll-like receptor-mediated IL-6 secretion by macrophage-like J774.1 cells. Biosci Biotechnol Biochem. 75(7):1283-9.
2. Kushida T. et al., 2011. Enhancement of Dectin-2 gene expression by lignin-carbohydrate complex from Lentinus edodes mycelia extract (LEM) in a mouse macrophage-like cell line. Anticancer Res. 31(4):1241-8.
3. Takeda Y. et al., 2007. Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells. Biomacromolecules. 8(4):1178-86.
4. Wilden H. et al., 2009. Expression of RIG-I, IRF3, IFN-beta and IRF7 determines resistance or susceptibility of cells to infection by Newcastle Disease Virus. Int J Oncol. 34(4):971-82.