iNOS Luciferase - (LUCPorter™) Stable Reporter Cell Line

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iNOS Luciferase - (LUCPorter™) Stable Reporter Cell Line Summary

• Description: The iNOS/LUCPoter(TM) reporter cell line is designed to monitor the induction of iNOS and can be used for screening of agonists, antagonists or signaling inhibitors of iNOS induction as well as for studying the iNOS induction-related signaling pathways.
  
  Contents: 3-4 x 10^6 cells
  Biosafety Level 2
• Immunogen: iNOS Luciferase - (LUCPorter (TM)) Stable Reporter Cell Line is a stably transfected RAW 264.7 cell line which expresses an optimized Renilla luciferase reporter gene (RenSP) under the transcriptional control of the iNOS promoter. Inducible nitric oxide synthase (iNOS) is an inducible enzyme that catalyzes the production of nitric oxide (NO) from L-arginine. the pathogenesis of septic shock. NO is one of the smallest signaling molecules that can diffuse into the cell and is involved in various physiological functions, pathogenesis of septic shock, many diseases associated with autoimmunity, and tumorigenesis. iNOS gene is generally known to be induced by various proinflammatory cytokines and pathogen-associated molecular patterns such as TLR ligands. As shown in Figure 1, the iNOS promoter activity in the iNOS/LUCPorter(TM) reporter cell line was induced by various TLR ligands (Figure 1).
• Selection Agent: Puromycin at 3 ug/ml
• Growth Properties: Adherent
  Morphology : Macrophage
• RCL Type: Luciferase - (LUCPorter™)
• Host: RAW264.7
• Gene: NOS2

Applications/Dilutions

• Dilutions:
  • Ligand Activation
• Application Notes: Ligand activation: See image for Induction of iNOS promoter activity by various TLR ligands and phorbol 12-myristate 13-acetate (PMA). The iNOS/LUCPorter(TM) RAW cell line was plated in 96-well white plates at 8.5 x 10^5 cells/well. After 16 h, cells were stimulated with 10 ng/ml Pam3CSK4 (TLR1/2 ligand; NBP2-25297), 10 ng/ml MALP-2 (TLR2/6 ligand; NBP2--26219), 40 ug/ml Poly(I:C) (TLR3 ligand; NBP2-25288), 0.5 ug/ml LPS (TLR4 ligand; NBP2-25295), 100 ng/ml Flagellin (TLR5 ligand; NBP2-25289), 10 ug/ml R848 (TLR7/8 ligand; NBP2-26231), 10 ug/ml mCpG (mouse TLR9 ligand; NBP2-26235) and 100 ng/ml PMA for 16 h. Luciferase activity was then analyzed by directly adding the complete mixture of luciferase reporter assay reagent (NBP2-25287) into each well of the plate. After 10 min, the plate was read in a plate luminometer.
• Readout System:
  • 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 (Note: The selection agents for this cell line is puromycin at 3 ug/ml).

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

• EC 1.14.13.39
• Hepatocyte NOS
• HEP-NOS
• Inducible NO synthase
• Inducible NOS
• iNOS
• nitric oxide synthase 2, inducible
• nitric oxide synthase 2A (inducible, hepatocytes)
• nitric oxide synthase, inducible
• nitric oxide synthase, macrophage
• NOS type II
• NOS
• NOS2
• NOS2A
• Peptidyl-cysteine S-nitrosylase NOS2

Background

Nitric oxide (NO) is a colorless, free radical gas that carries a variety of messages between cells. Vasorelaxation, neurotransmission and cytotoxicity can all be potentiated through cellular response to NO. NO production is mediated by members of the nitric oxide synthase (NOS) family including the two constitutive isoforms: brain, bNOS, or neuronal NOS, nNOS (type I) and endothelial cell NOS, eNOS (type III); along with the inducible isoform, iNOS (type II). NOS catalyzes the oxidization of L-arginine to produce L-citrulline and NO, requiring the cofactors calmodulin, nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN), heme, and tetrahydrobiopterin (1).

The 131 kDa enzyme, iNOS, is found in a variety of cell types including macrophages, hepatocytes, synoviocytes, and smooth muscle cells. While constitutively expressed in kidneys, in other tissues iNOS is induced by bacterial lipopolysaccharides (LPS), growth factors, and cytokines such as IFN-gamma, TNF, IL-1 and IL-2. iNOS is not regulated by the level of intracellular Ca2+ and is constantly active as a dimer when expressed. iNOS activity is elevated in a variety of diseases including atherosclerosis, heart failure, sepsis, solid tumors, and type 2 diabetes. Acting as a critical mediator of inflammation and apoptosis, iNOS inhibitors have been shown to alleviate obesity and stress inducted insulin resistance in mouse models (2,3).

References

1. Forstermann U, and Sessa WC. (2012) Nitric oxide synthases: regulation and function. Eur Heart J. 33(7): 829-837. PMID: 21890489

2. Aktan F. (2004) iNOS-mediated nitric oxide production and its regulation. Life Sci. 75(6):639-53. PMID: 15172174

3. Cinelli MA, Do HT, Miley GP, Silverman RB. (2020) Inducible nitric oxide synthase: Regulation, structure, and inhibition. Med Res Rev. 40(1):158-189. PMID: 31192483

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.

FAQs for iNOS Reporter Cell Line (NBP2-32782). (Showing 1 - 1 of 1 FAQ).

1. We need to evaluate the level of iNOS in Rat sciatic nerve by Western Blot. I see that you have few antibodies suitable for that, could you please recommend the best what you have for our purposes?
   • We have 4 iNOS antibodies that are suitable for your experiment. The best choice for you will depend on your specific preferences, in this case the main difference will be the immunogen location. If the immunogen is not important to you, then I would recommend choosing NB300-605 as it gives you the best value and we are able to provide the exact immunogen.

Bioinformatics

• Gene Symbol: NOS2