iNOS Activation Kit

The kit contains a monoclonal mouse anti-iNOS antibody, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore, and the various other reagents and buffers required for immunofluorescence labeling of iNOS for high content screening (HCS) assays.

This assay is based on the elicitation of the immune response. After stimulation with lipopolysaccharide (LPS) and interferon gamma (IFNγ), or other harmful stimuli, immune cells produce cytokines, chemokines and oxidative radicals as a defensive mechanism. Nitric oxide synthase (NOS) is responsible for generation of nitric oxide (NO) through oxidizing L-arginine to L-citrulline. Nitric oxide acts as a signaling molecule in the cardiovascular system, as well as an inflammatory molecule and can react with other species to form more potent radicals such as peroxynitrite and lipid peroxides. There are three forms of NOS: eNOS expressed in endothelial cells; nNOS, expressed in neuronal cells and iNOS, expressed in macrophages. Induction of iNOS through inflammatory mediators results in protein nitration, apoptosis induction, oxidative stress, DNA damage and respiration inhibition.

LPS and IFNγ induction of iNOS in RAW 264.7 murine macrophages was quantitatively assayed using the iNOS Activation Kits, the Cellomics ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

 
Figure 2. iNOS induction after treatment with LPS and interferon gamma (IFNγ). RAW 264.7 murine macrophages were challenged with 500 ng/ml LPS and 100 ng/ml mouse interferon gamma overnight (20-24 hours) to measure cytoplasmic induction of iNOS. Cells were then fixed and stained with anti-iNOS primary antibody and DyLight 549 Goat anti-Mouse secondary antibody (orange). Cells were also labeled with Hoechst Dye for nuclear staining (blue). Untreated cells (left); treated cells (right); dose response curves (bottom). EC₅₀ = 90 ± 0.02 ng/ml LPS and 100 ± 0.02 ng/ml IFNγ.

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