NETosis Assay Kit #41855
Product Information
Storage
All components in this kit are stable for at least 12 months when stored at the recommended temperature.
Protocol
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).
- Thiam, H.R. et al. (2020) Annu Rev Cell Dev Biol 36, 191-218.
- Brinkmann, V. et al. (2004) Science 303, 1532-5.
- Li, P. et al. (2010) J Exp Med 207, 1853-62.
- Wong, S.L. and Wagner, D.D. (2018) FASEB J, fj201800691R.
- Papayannopoulos, V. et al. (2010) J Cell Biol 191, 677-91.
- Metzler, K.D. et al. (2014) Cell Rep 8, 883-96.
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