Human TLR4 Stable Cell Line

Product Discontinued

Human TLR4 Stable Cell Line Summary

• Description: The TLR4 stable cell line can be used for TLR4 flow cytometric calibration and detection control as well as TLR4-dependent functional assays. TLR4 expression in this stable cell line has been validated by Western blotting (Fig. 1) and flow cytometry (Fig. 2). Functional activity of this stable cell line has been validated by the NF-kB/SEAPorte™ Assay Kit (NBP2-25286, Fig. 3).
  
  Contents: 3~4 x 10^6 cells
  Biosafety Level: 2
• Immunogen: The TLR4 stable cell line is a stably transfected cell line which expresses full-length human Toll-like receptor 4 (TLR4) with an N-terminal HA tag.
• Target Species: Human
• Specificity: TLR4
• Selection Agent: Blasticidin
• Growth Properties: Adherent Morphology: Epithelial
• RCL Type: Stable Cell Line
• Host: HEK293
• Gene: TLR4

Applications/Dilutions

• Dilutions:
  • Functional
• Application Notes: Use in Functional reported in scientific literature (PMID26248657)
• Readout System:
  • NFkB Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric) NBP2-25286
  • Cell Surface Staining Flow Cytometry Kit NBP2-26247

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 + 10 ug/ml blasticidin.

Details for Array

• Type: Cell

Notes

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 stable line.
- Wash hands after handling the stable line and before leaving the lab.
- Decontaminate work surface with disinfectant or 70% ethanol before and after working with cells.
- All waste should be considered hazardous.
- Dispose of all liquid waste after each experiment and treat with bleach.

Alternate Names for Human TLR4 Stable 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.

Publications for TLR4 Reporter Cell Line (NBP2-26268)(3)

Publications using NBP2-26268

• Lee SU, Oh ES, Ryu HW et al. Longifolioside A inhibits TLR4-mediated inflammatory responses by blocking PKC delta activation in LPS-stimulated THP-1 macrophages Cytokine 2020-01-01 [PMID: 32388485]
• O'Neill S, Humphries D, Tse G et al. Heat shock protein 90 inhibition abrogates TLR4-mediated NF-kB activity and reduces renal ischemia-reperfusion injury. Sci Rep. 2015-08-07 [PMID: 26248657] (Func)
• Mukherjee S, Biswas R, Biswas T. Alternative TLRs are stimulated by bacterial ligand to induce TLR2-unresponsive colon cell response. Cell Signal. 2013-08-01 [PMID: 23665374] (WB)
  
  Details:
  TLR Stable cell lines cited in Fig 2C for IP/WB:1. TLR1 Stable Cell Line (IML-201)2. TLR2 Stable Cell Line (IML-202)3. TLR4 Stable Cell Line (IML-204)Note: The cell lines were exposed to porin and IP'd with a TLR1, TLR2, or TLR4 antibody, respectively, fo

FAQs for TLR4 Reporter Cell Line (NBP2-26268). (Showing 1 - 2 of 2 FAQs).

1. The customer stimulated the products TLR4 Stable Cell Line (NBP2-26268) and TLR2 Stable Cell Line (NBP2-26266) with their ligand, and he detected the expression of Defencin by semi-quantitative PCR. Defencin is the response factor downstream of TLR signaling. However the expression level is same to normal HEK cell. When he also used HaCat cell, the expression level of Defencin increased commensurately. He suggests the HaCat cell line seems to be better response than these products. Do you know why this is? Also, TLR2 exists in complex with TLR1 or TLR6 in native condition. TLR4 exists in complex with MD2 and CD14. He noticed whether the TLR in these cell line are not over-expressd, because these cell lines does not over-express their complex element together. He would like to make sure that the expression level of TLR in IMGENEX TLR cell line is higher than native cell line. Additionally he wants to know whether TLR can be detected by using TLR antibody in western blotting generally. Tag antibody can be not used for such analysis, because Tag antibody can not detect native endogenous TLR. Coud you please tell us your suggestion?
   • Functional activity of this cell line requires co-transfection of MD-2 and CD14. With regards to readout assays, we have evaluated both the NBP2-26268 (TLR4) and NBP2-26266 (TLR2) with the SEAP reporter assays as shown on the respective data sheets. We don't perform semi-quantitative PCR and I am sorry we don't have any information regarding the ligand the customer used nor a Defencin PCR quantitative PCR readout assay. Hence, it would be difficult to comment further on the customer's results. We would suggest that the customer use the techniques and assays shown on the data sheet as positive controls for cell line function. This includes the SEAP reporter assays which require co-transfection of the NF-kB SEAP reporter, and for the TLR4 cell it also requires co-transfection of MD2 and CD14. This also includes using established TLR4 (LPS) and TLR2 (PAM3CSK4). Once the researcher establishes the positive control system, they can move to testing their ligand with the SEAP readout assay and then to their Defencin readout assay. However, it must be recognized that the suitability of the cell lines for a researcher's model system needs to be empirically determined and optimized by the researcher. Both the NBP2-26266 and NBP2-26268 cell lines are selected for function as assayed by a SEAP reporter assay (see data on data sheets). Overexpression results are shown by flow cytometry using the appropriate TLR antibodies with respect to the Vector control cell line. Please refer to the data sheets for these results. For WB, we use an HA Tag antibody. The TLRs were not recognized by WB using the TLR antibodies we evaluated. We would suggest using a Tag antibody if the researcher wishes to detect the TLR constructs expressed by the cell lines. We are not studying endogenous TLRs in the cell lines, the focus is on the functional aspects of the TLR genes stably transfected into the cell lines. If the customer wishes to use TLR antibodies to assess the TLRs, then flow cytometry assays should be performed as shown on the data sheets. On another note, HEK293 do not express functional TLR2 or TLR4. I am not sure what ligand the researcher is using but activation of normal HEK293 cells might be occurring through other pathways.
2. Does the TLR4 cell line express the related factors (MD2, CD14 and LBP). Has expression of each gene been confirmed?
   • We have not evaluated the gene expression of MD2, CD14 or LBP in this cell line. The parent cell line is HEK293 as denoted in the data sheet. One would expect that the gene expression profile would be similar to the HEK293 parent cell line. There is considerable information in the scientific literature about HEK293. We have not evaluated the gene expression profile of the parent HEK293 cell line in our hands.

Bioinformatics

• Gene Symbol: TLR4