NETosis Assay Kit (#41855) Datasheet Without Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Information

Storage

All components in this kit are stable for at least 12 months when stored at the recommended temperature.

Product Description

The NETosis Assay Kit is a quantitative colorimetric assay that can be used to study the process of NETosis. In this kit, primary neutrophils are stimulated with PMA (included in the kit) to release neutrophil extracellular traps (NETs), leading to the production of neutrophil elastase. The unbound neutrophil elastase will be washed away, leaving behind NETs with bound neutrophil elastase. The S7 nuclease is used to digest NET DNA, releasing the neutrophil elastase from the NETs. When the neutrophil elastase substrate is added to the samples, the substrate is selectively cleaved by the neutrophil elastase to produce 4-nitroaniline that absorbs light at 405 nm. The NETosis Assay Kit does not depend upon the DNA component of NETs, as DNA release can occur independently of NETosis. The kit provides enough reagents to test up to 80 individual samples with 16 standard curve wells in a 96-well plate, or 24 samples in duplicate in a 24-well plate.

Background

NETosis is a unique form of regulated cell death that is characterized by membrane rupture and the extrusion of chromatin, histones, and granular and cytoplasmic components into a web-like structure called neutrophil extracellular traps (NETs) (reviewed in 1). NETosis has been associated with host defense to pathogens as well as a number of disease states, including autoimmune diseases, thrombosis, cardiovascular diseases, and tumor progression. NETosis was identified as a response to bacterial infection and can be activated by lipopolysaccharide (LPS) as well as inflammatory pathway activators like phorbol-12-myristate-13-acetate (PMA) (2). It can occur via multiple pathways, but several key players have emerged. The calcium-dependent enzyme protein-arginine deiminase 4 (PAD4) catalyzes hypercitrullination of histones that contributes to chromatin decondensation (3,4). In addition, activation of proteases, including neutrophil elastase (ELANE), myeloperoxidase (MPO), and Cathepsin G, leads to impairment of cytoskeletal structures and degradation of histones during NETosis (5,6).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

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