Invivogen
Menu

RAW-Dual™ Cells

Product Unit size Cat. code Docs. Qty. Price

RAW-Dual™ Cells

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

Show product

3-7 x 10e6 cells

rawd-ismip
+-
$1,457

Notification:  This product is for internal research use only. Additional rights may be available. Please visit InvivoGen’s Terms and Conditions.

Dual IRF-Lucia and MIP-2-SEAP murine macrophage reporter cell line

RAW Dual pathway
TLR and Type I IFN signaling in RAW-Dual™ Cells

RAW-Dual(IRF-Lucia/KI-[MIP-2]SEAP) cells were generated from RAW 264.7 murine macrophages, which express many pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) TLR2 and TLR4 [1], the cytosolic DNA sensor (CDS) cGAS [2], and the cyclic dinucleotide (CDN) sensor STING [2].

RAW‑Dual™ cells stably express two reporter genes encoding SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase.

SEAP expression depends on the activation of the endogenous MIP-2 promoter. MIP‑2, also known as CXCL2, is the murine homolog of human IL-8, a chemokine produced in an NF-κB-dependent manner [3]. The MIP-2 ORF has been replaced by the SEAP ORF using knockin technology. Hence SEAP expression reports activation of NF-κB.

The Lucia luciferase gene is under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements (ISRE). It reports the activation of interferon regulatory factors (IRFs).

Both reporter proteins are secreted and readily measurable in the cell culture supernatant using  QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia,  a Lucia and Gaussia luciferase detection reagent. Alternatively, SEAP activity can be detected using HEK-Blue™ Detection, a cell culture medium allowing real-time detection of SEAP.

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

RAW-DualCells are resistant to Zeocin®.

 

References:

1. Underhill DM. et al., 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 401:811-5.
2. Hornef MW. et al., 2003. Intracellular Recognition of Lipopolysaccharide by Toll-like Receptor 4 in Intestinal Epithelial Cells. J Exp Med.198:1225-35.
3. Tanaka Y. & Chen ZJ., 2012. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal. 5(214):ra20.
4. 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.

Figures

IRF INDUCTION (Lucia luciferase reporter)
IRF INDUCTION (Lucia luciferase reporter)

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%).

MIP-2 (NF-kB) INDUCTION (SEAP reporter)
MIP-2 (NF-kB) INDUCTION (SEAP reporter)

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. 

Back to the top

Specifications

Antibiotic resistance: Zeocin®

Quality Control:

  • The biallelic replacement of the mouse MIP-2 (also known as CXCL2) gene with the SEAP reporter gene was verified by PCR and sequencing.
    The inability to produce MIP-2 has been confirmed by ELISA.
  • Reporter activity has been verified by functional assays.
  • Cell line stability for 20 passages following thawing has been verified.
  • RAW-Dual™ cells are guaranteed mycoplasma-free.
     

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

Back to the top

Contents

  • 3-7 x 106 of RAW-Dual™ cells in a cryovial or shipping flask
  • 1 ml Zeocin® (100 mg/ml)
  • 1 ml 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)

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

Back to the top

Citations

Load more
Customer Service
& Technical Support
Shopping cart is empty