TLR4 KO Reporter RAW 264.7 Cells
Product | Unit size | Cat. code | Docs. | Qty. | Price | |
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RAW-Dual™ KO-TLR4 Cells TLR4 knockout IRF and MIP-2 (NF-κB) reporter mouse macrophages |
Show product |
3-7 x 10e6 cells |
rawd-kotlr4
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TLR4 knockout IRF-Lucia & MIP-2-knockin-SEAP murine macrophage reporter cell line
TLR and Type I IFN signaling in RAW-Dual™ KO-TLR4 Cells
RAW-Dual™ KO-TLR4 (IRF-Lucia/KI-[MIP-2]SEAP) cells were generated from RAW-Dual™ cells through stable gene knockout of Toll‑like receptor 4 (TLR4). These cells derive from RAW 264.7 murine macrophages which express many pattern recognition receptors (PRRs) [1].
RAW‑Dual™ KO-TLR4, along with their parental cell line RAW-Dual™ cells, can be used to:
- Investigate TLR4 signaling
- Study TLR4-independent biological activity of test compounds.
Both cell lines 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 is a chemokine produced in an NF-κB-dependent manner [2]. The MIP-2 ORF has been replaced by the SEAP ORF using knockin technology.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 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™ KO-TLR4 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.
As expected, RAW-Dual™ KO-TLR4 cells do not respond to TLR4 agonists such as whole lipopolysaccharide (LPS) or monophosphoryl lipid A (MLPA).
RAW-Dual KO-TLR4™ cells are resistant to Zeocin®.
References:
1. West A. et al., 2011.TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472:476-80.
2. Kim D. et al., 2003. NF-kappaB and c-Jun-dependent regulation of macrophage inflammatory protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells. Mol Immunol. 40:633-43.
Specifications
Antibiotic resistance: Zeocin®
Growth medium: DMEM, 2 mM L-glutamine, 4.5 g/l glucose, 10% FBS supplemented with 100 µg/ml Normocin™
Quality Control:
- Biallelic TLR4 knockout has been verified by DNA sequencing and functional assays.
- The biallelic replacement of the mouse MIP-2 (macrophage inflammatory protein-2; CXCL2; murine homolog of IL-8) open reading frame (ORF) with the SEAP reporter ORF has been verified by PCR and sequencing. Furthermore, the inability to produce MIP-2 has been confirmed by ELISA.
- Activity of RAW-Dual™ KO-TLR4 cells has been tested with pattern recognition receptor (PRR) ligands that trigger the NF-κB and interferon regulatory factor (IRF) signaling pathways.
- Cell line stability for 20 passages following thawing has been verified.
- RAW-Dual™ KO-TLR4 cells are guaranteed mycoplasma-free.
This product is covered by a Limited Use License (See Terms and Conditions).
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- 3-7 x 106 of RAW-Dual™ KO-TLR4 cells in a cryovial or shipping flask
- 1 ml of Zeocin® (100 mg/ml)
- 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi.
- 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)
Shipped on dry ice (Europe, USA, Canada and some areas in Asia)
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Toll‑like receptor 4 (TLR4) is the receptor for Gram-negative lipopolysaccharide (LPS) and its toxic moiety called lipid A. TLR4 interacts with three different extracellular proteins: the LPS-binding protein (LBP), CD14, and the myeloid differentiation protein 2 (MD‑2). Their interaction induces a signaling cascade resulting in the activation of NF-κB and the production of proinflammatory cytokines. LPS contamination is a common issue arising when testing novel compounds, such as recombinant proteins, leading to unreliable results. TLR4 knockout cells ensure the study of PRR pathways without interference from LPS.
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