RAW-Dual™ Cells

RAW-Dual™ Cells Unit size Cat. code Docs Qty Price
Dual IRF and MIP-2 (NF-kB) reporter mouse macrophages
3-7 x 10e6 cells

Dual IRF and MIP-2 (NF-kB) reporter mouse macrophages

RAW-Dual(IRF-Lucia/KIN-[MIP-2]SEAP) cells are derived from RAW 264.7 macrophages, a murine immune cell model that expresses many PRRs such as the Toll-like receptors (TLRs) TLR2 [1] and TLR4 [2], and the cyclic dinucleotide sensor STING [3].

RAW-Dual™ cells respond to PRR ligands that trigger the NF-kB and the IRF pathways. They also respond to murine, but not human, IFN-α and IFN-β.

These cells stably express two different secreted reporter genes: the SEAP (secreted embryonic alkaline phosphatase) gene and the Lucia luciferase gene, which encodes a novel secreted luciferase.

The Lucia luciferase gene is under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements.

The SEAP gene expression depends on the activation of the MIP-2 promoter. MIP-2, the murine ortholog of IL-8, is a chemokine which is produced in an NF-kB dependent-manner [4].

The MIP-2 ORF has been replaced by the SEAP ORF, while the MIP-2 promoter has been left intact; thus, these cells produce SEAP upon activation of the endogenous MIP-2 promoter but do not produce MIP-2.

Both reporter proteins are readily measurable in the cell culture supernatant by using QUANTI-Blue™, a SEAP detection reagent, and QUANTI-Luc™, a Lucia luciferase detection reagent.

As a result, RAW-Dual™ cells allow to simultaneously study the NF-kB pathway, by assessing the activity of SEAP, and the IRF pathway, by monitoring the activity of Lucia luciferase.

RAW-DualCells are resistant to  Zeocin™.

Underhill DM. et al., 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 401:811-5.
Hornef MW. et al., 2003. Intracellular Recognition of Lipopolysaccharide by Toll-like Receptor 4 in Intestinal Epithelial Cells. J Exp Med.198:1225-35..
Tanaka Y. & Chen ZJ., 2012. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal. 5(214):ra20.
Kim DS. et al., 2003. NF-kappaB and c-Jundependent regulation of macrophage inflammatory protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells. Mol Immunol. 40(9):633-43.

RAW-Dual™ cells were stimulated with murine IFN-β (mIFN-β; 1x104 U/ml), mIFN-α (1x104 U/ml), LPS-EK Ultrapure (100 ng/ml; TLR ligand), poly(I:C) HMW (1 μg/ml; TLR3 ligand), Poly(I:C) HMW/LyoVec™ (1 μg/ml; RIG-I/MDA5 ligand), poly(dA:dT)/LyoVec™ (1 μg/ml; RIG-I ligand & cytosolic DNA sensor (CDS) ligand), VACV-70/LyoVec™ (1 μg/ml; CDS ligand), and 2’3’-cGAMP (10 μg/ml; STING ligand). 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 IRF induction of each ligand is expressed relative to that of mIFN-β at 1 x 104 U/ml (taken as 100%).

RAW-Dual™ cells were incubated with HKLM (1x108 cells/ml; heat-killed Listeria monocytogenes, a TLR2 ligand), Pam3CSK4 (1 μg/ml; TLR2 ligand), poly(I:C) HMW (10 μg/ml; TLR3 ligand), LPS-EK Ultrapure (1 μg/ml; TLR4 ligand), FLA-ST (10 μg/ml; TLR5 ligand), CL075 (1 μg/ml; TLR 7/8 ligand), ODN 1826 (1 μg/ml; TLR9 ligand), Zymosan (10 μg/ml; TLR2 & Dectin-1 ligand), TDB (100 μg/ml; Mincle ligand). Non-induced cells (NI) have been included as a negative control. After a 24h incubation, NF-kB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. 

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The biallelic replacement of the mouse MIP-2 (also known as CXCL2) gene with the SEAP reporter gene was verified by PCR and sequencing. Furthermore, the inability to produce MIP-2 has been confirmed by ELISA.

Activity of RAW-Dualcells was tested with pattern recognition receptor (PRR) ligands that trigger the NF-kB and interferon regulatory factor (IRF) signaling pathways.

The stability of this cell line for 20 passages following thawing has been verified.

Antibiotic resistance: Zeocin™

Guaranteed mycoplasma-free.

Growth medium: DMEM, 2 mM L-glutamine, 4.5 g/l glucose, 10% FBS supplemented with 100 µg/ml Normocin™ .

This product is covered by a Limited Use License (See Terms and Conditions).

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Shipped on dry ice

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RAW-Dual pathway

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