Human TLR4 Luciferase - (LUCPorter™) Stable Reporter Cell Line

Product Discontinued

Human TLR4 Luciferase - (LUCPorter™) Stable Reporter Cell Line Summary

• Description: The TLR4/MD-2/CD14/IL-8 Prom/LUCPorter™ reporter cell line can be used for TLR4 signaling dependent functional assays as well as screening of TLR4 agonists, antagonists or signaling inhibitors. This cell line is also suitable for testing neutralizing antibodies for TLR4, MD-2 or CD14. The cell line has been validated by ligand dose response assay, DMSO tolerance and an antagonist assay using the TLR2 / TLR4 inhibitor peptide (COBRA) (Figures 1 through 3).
  
  Contents: 3~4 x 10^6 cells
  Biosafety Level: 2
• Immunogen: The TLR4/MD-2/CD14/IL-8 Prom/LUCPorter™ reporter cell line is a stably transfected HeLa cell line which expresses human TLR4, MD-2 and CD14 as well as an optimized Renilla luciferase reporter gene (RenSP) under the transcriptional control of the IL-8 promoter. The IL-8 promoter has been utilized since IL-8 is one of the major pro-inflammatory cytokines induced by ligand (such as LPS)-mediated TLR4 activation.
• Target Species: Human
• Specificity: TLR4/MD-2/CD14/IL-8 Prom/LUCPorter™
• Selection Agent: TLR4 cell line is puromycin, blasticidin and G418.
• Growth Properties: Morphology: Epithelial-like and growth properties is Adherent
• RCL Type: Luciferase - (LUCPorter™)
• Reporter Gene: Renilla luciferase
• Host: HeLa
• Gene: TLR4

Applications/Dilutions

• Dilutions:
  • Ligand Activation
• Agonist:
  • LPS from E. Coli, TLR4 ligand NBP2-25295
• Antagonist:
  • TLR4 Inhibitor Peptide Set NBP2-26244
  • TIRAP (TLR2 and TLR4) Inhibitor Peptide Set NBP2-26245
• Readout System:
  • LightSwitch Luciferase Assay Reagent NBP2-25287
  • LightSwitch Luciferase Assay Reagent NBP2-32066

Packaging, Storage & Formulations

• Storage: Store in gas phase of liquid nitrogen.
• Reconstitution Instructions: Complete Growth Medium: DMEM with 4.5 g/L glucose + 10% FBS + 4 mM L-glutamine + 1 mM sodium pyruvate + 100 units/ml penicillin + 0.1 mg/ml streptomycin + 1 ug/ml puromycin + 5 ug/ml blasticidin + 1.0 mg/ml G418 (Geneticin).

Details for Array

• Type: Cell

Notes

The HeLa cells used to generate this reporter line is an immortal human cell line. Any human lines should be considered as biohazard and be used in a Biosafety II laboratory. Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should be observed.
- Use pipette aids to prevent ingestion and keep aerosols down to a minimum.
- No eating, drinking or smoking while handling the TLR4 line.
- Wash hands after handling the TLR4 line and before leaving the lab.
- Decontaminate work surface with disinfectant or 70% ethanol before and after working with the cell line.
- All waste should be considered hazardous.
- Dispose of all liquid waste after each experiment and treat with bleach.

Alternate Names for Human TLR4 Luciferase - (LUCPorter™) Stable Reporter Cell Line

• ARMD10
• CD_antigen: CD284
• CD284 antigen
• CD284
• EC 3.2.2.6
• EC:3.2.2.6
• homolog of Drosophila toll
• hToll
• TLR4
• TLR-4
• toll like receptor 4 protein
• TOLL
• toll-like receptor 4

Background

TLR4 (Toll-like receptor 4) is a type-1 transmembrane glycoprotein that is a pattern recognition receptor (PRR) belonging to the TLR family (1-3). TLR4 is expressed in many tissues and is most abundantly expressed in the placenta, spleen, and peripheral blood leukocytes (1). Human TLR4 is synthesized as a 839 amino acid (aa) protein containing a signal sequence (1-23 aa), an extracellular domain (ECD) (24-631 aa), a transmembrane domain (632-652 aa), and Toll/interleukin-1 receptor (TIR) cytoplasmic domain (652-839 aa) with a theoretical molecular weight of 95 kDa (3, 4). The ECD contains 21 leucine-rich repeats (LRRs) and has a horseshoe-shaped structure (3, 4). TLR4 requires binding with the co-receptor myeloid differentiation protein 2 (MD2) largely via hydrophilic interactions for proper ligand sensing and signaling (2-4). In general, the TLR family plays a role in activation of innate immunity and responds to a variety of pathogen-associated molecular patterns (PAMPs) (5). TLR4 is specifically responsive to lipopolysaccharide (LPS), which is found on the outer-membrane of most ram-negative bacteria (3-5). Activation of TLR4 requires binding of a ligand, such as LPS to MD2, followed by MD2-LPS complex binding to TLR4, resulting in a partial complex (TLR4-MD2/LPS) (3, 5). To become fully active, two partial complexes must dimerize thereby allowing the TIR domains of TLR4 to bind other adapter molecular and initiate signaling, triggering an inflammatory response and cytokine production (3, 5).

TLR4 signaling occurs through two distinct pathways: The MyD88 (myeloid differentiation primary response gene 88)-dependent pathway and the MyD88-independent (TRIF-dependent, TIR domain-containing adaptor inducing IFN-beta) pathway (3, 5-7). The MyD88-dependent pathway occurs mainly at the plasma membrane and involves the binding of MyD88-adaptor-like (MAL) protein followed by a signaling cascade that results in the activation of transcription factors including nuclear factor-kappaB (NF-kappaB) that promote the secretion of inflammatory molecules and increased phagocytosis (5-7). Conversely, the MyD88-independent pathway occurs after TLR4-MD2 complex internalization in the endosomal compartment. This pathway involves the binding of adapter proteins TRIF and TRIF-related adaptor molecule (TRAM), a signaling activation cascade resulting in IFN regulatory factor 3 (IRF3) translocation into the nucleus, and secretion of interferon-beta (INF-beta) genes and increased phagocytosis (5-7).

Given its expression on immune-related cells and its role in inflammation, TLR4 activation can contribute to various diseases (6-8). For instance, several studies have found that TLR4 activation is associated with neurodegeneration and several central nervous system (CNS) pathologies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (6, 7). Furthermore, TLR4 mutations have been shown to lead to higher rates of infections and increased susceptibility to sepsis (7-8). One potential therapeutic approach aimed at targeting TLR4 and neuroinflammation is polyphenolic compounds which include flavonoids and phenolic acids and alcohols (8).

Alternative names for TLR4 includes 76B357.1, ARMD10, CD284 antigen, CD284, EC 3.2.2.6, homolog of Drosophila toll, hToll, toll like receptor 4 protein, TOLL, toll-like receptor 4.

References

1. Vaure, C., & Liu, Y. (2014). A comparative review of toll-like receptor 4 expression and functionality in different animal species. Frontiers in immunology. https://doi.org/10.3389/fimmu.2014.00316

2. Park, B. S., & Lee, J. O. (2013). Recognition of lipopolysaccharide pattern by TLR4 complexes. Experimental & molecular medicine. https://doi.org/10.1038/emm.2013.97

3. Krishnan, J., Anwar, M.A., & Choi, S. (2016) TLR4 (Toll-Like Receptor 4). In: Choi S. (eds) Encyclopedia of Signaling Molecules. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-6438-9_592-1

4. Botos, I., Segal, D. M., & Davies, D. R. (2011). The structural biology of Toll-like receptors. Structure. https://doi.org/10.1016/j.str.2011.02.004

5. Lu, Y. C., Yeh, W. C., & Ohashi, P. S. (2008). LPS/TLR4 signal transduction pathway. Cytokine. https://doi.org/10.1016/j.cyto.2008.01.006

6. Leitner, G. R., Wenzel, T. J., Marshall, N., Gates, E. J., & Klegeris, A. (2019). Targeting toll-like receptor 4 to modulate neuroinflammation in central nervous system disorders. Expert opinion on therapeutic targets. https://doi.org/10.1080/14728222.2019.1676416

7. Molteni, M., Gemma, S., & Rossetti, C. (2016). The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation. Mediators of inflammation. https://doi.org/10.1155/2016/6978936

8. Rahimifard, M., Maqbool, F., Moeini-Nodeh, S., Niaz, K., Abdollahi, M., Braidy, N., Nabavi, S. M., & Nabavi, S. F. (2017). Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation. Ageing research reviews. https://doi.org/10.1016/j.arr.2017.02.004

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Reporter Cell Lines are guaranteed for 1 year from date of receipt.

Bioinformatics

• Gene Symbol: TLR4