Human TLR4/MD-2/CD14 SEAP - (SEAPorter™) Stable Reporter Cell Line

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Human TLR4/MD-2/CD14 SEAP - (SEAPorter™) Stable Reporter Cell Line Summary

• Description: The TLR4/MD-2/CD14 reporter line can be used for TLR4-dependent functional assays as well as screening of TLR4 agonists or antagonists. The TLR4/MD-2/CD14 reporter cell line has been validated by a ligand dose response assay (Figure 1).
  
  Contents: 3~4 x 10^6 cells
  Biosafety Level: 2
• Immunogen: The TLR4/MD-2/CD14 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor 4 (TLR4), human MD-2, human CD14 and secreted alkaline phosphatase (SEAP) reporter genes under the transcriptional control of an NF-kB response element.
• Target Species: Human
• Specificity: TLR4/MD-2/CD14/NF-kB/SEAP
• Selection Agent: Blasticidin, puromycin, zeocin and G418
• Growth Properties: Adherent Morphology: Epithelial
• RCL Type: SEAP - (SEAPorter™)
• Host: HEK293

Applications/Dilutions

• Dilutions:
  • Functional
  • In vitro assay
  • Ligand Activation
• Application Notes: Use in function and in-vitro reported in scientific literature (PMID 25640528)
• Agonist:
  • LPS from E. Coli, TLR4 ligand NBP2-26216
  • LPS from E. Coli, TLR4 ligand NBP2-25295
• Readout System:
  • Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric) NBP2-25285

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 + 2 ug/ml puromycin + 0.2 mg/ml zeocin + 0.5 mg/ml G418 (Geneticin).

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 TLR4/MD-2/CD14 line.
- Wash hands after handling the TLR4/MD-2/CD14 line and before leaving the lab.
- Decontaminate work surface with disinfectant or 70% ethanol before and after working with the TLR4/MD-2/CD14 reporter line.
- All waste should be considered hazardous.
- Dispose of all liquid waste after each experiment and treat with bleach

Alternate Names for Human TLR4/MD-2/CD14 SEAP - (SEAPorter™) Stable Reporter Cell Line

• TLR4,MD2,CD14

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/MD-2/CD14 Reporter Cell Line (NBP2-26503)(6)

Publications using NBP2-26503

• Yamaguchi M, Hirose Y, Takemura M, et al Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA. Front Cell Infect Microbiol. 2019-08-20 [PMID: 31482074] (Bacteria)
  
  Details:
  Mice were infected with S. pneumoniae.
• Ryan DA, Degardin M, Alam S et al. Rational Design of Peptide Derivatives for Inhibition of MyD88-Mediated TLR Signaling in Human PBMCs and Epithelial Cells Exposed to F. tularensis. Chem Biol Drug Des. 2017-06-09 [PMID: 28599094] (Biacore Binding Assay, Human)
• McKim JM, Wilga PC, Pregenzer JF, Blakemore WR. The common food additive carrageenan is not a ligand for toll-like- receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model Food Chem. Toxicol. 2015-01-29 [PMID: 25640528] (Func, In vitro)
  
  Details:
  Human TLR4/MD-2/CD14 SEAP - (SEAPorterTM) Stable Reporter Cell Line [HEK293 cells stably transfected with a TLR4/MD-2/CD14/NF-kB/SEAP reporter construct - expresses full-length human TLR4, MD-2, CD14, and secreted alkaline phosphatase/SEAP under the control of an NFkB response element] used for evaluating the ability of different types of Carrageenan/CGN to bind and activate TLR4 signaling. SEAP was measured as an indicator of TLR4 activation with SEAPorterTM assay kit and CGN was found to not to activate the TLR4.
• Chan M, Hayashi T, Mathewson RD et al. Identification of substituted pyrimido[5,4-b]indoles as selective Toll-like receptor 4 ligands. J Med Chem. 2013-06-13 [PMID: 23656327]
• Feng D, Sangster-Guity N, Stone R et al. Differential requirement of histone acetylase and deacetylase activities for IRF5-mediated proinflammatory cytokine expression. J Immunol. 2010-11-15 [PMID: 20935208]
• Greene CM, Carroll TP, Smith SG et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005-02-01 [PMID: 15661927] (Func)
  
  Details:
  Functional Assays: 1.TLR2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-102). 2. TLR4/MD-2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104MD2). 3. TLR4/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104).4.TLR7/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-107). 5. LPS from E. Coli, TLR4 Ligand (IMG-2204). 6. SEAPorter Assay Kit (10055K).

FAQs for TLR4/MD-2/CD14 Reporter Cell Line (NBP2-26503). (Showing 1 - 2 of 2 FAQs).

1. Do the cell lines carry plasmids and is this why the selection agents are essential to grow the cells. Can the cells expand without losing their plasmids in the absence of selection agents? So what all plasmids are there for what all genes and what is the selection agent for the respective plasmids. Is SEAP also on plasmids?
   • This cell line contains 4 plasmids; TLR4, MD-2, CD14, and NF-kB-SEAP. This is why selection agents are essential when using this cell line. The 4 plasmids are selected using blasticidin, puromycin, zeocin and G418/Geneticin, respectively. SEAP is coupled to NFkB on a plasmid. I would encourage you to use the protocol for this product when growing your cells.
2. Are there any normalization techniques that can be used in an assay to make the results more reliable and remove the variability of cell numbers?
   • I would recommend that you do not change the media during the whole cell assay. The optimal volume of ligand solution should not exceed 10% of the media volume of each well. In addition, I would recommend that you omit the selection agents during the whole cell assay. The selection agents are required only for cell maintanence.