Neutrophil Extracellular Trap (NET) Assay Kit

Production of neutrophil extracellular traps (NETs) is a key feature of the neutrophil antimicrobial response in which neutrophils weave web-like nets from DNA, histones, and antimicrobial macromolecules upon encountering a bacterial challenge. These NETS, which are also comprised of myeloperoxidase, neutrophil elastase, and lactotransferrin, are extruded to capture and possibly kill any nearby bacteria. Once released, NETs are rapidly cleared by the action of plasma DNAse and by macrophages via scavenger receptor-mediated phagocytosis of the NET/neutrophil complex. Previous methods for detection of NET generation have used fluorescent DNA-intercalating dyes. However, because DNA release can also occur as a result of mechanical cellular injury or necrosis, the use of DNA as a definitive readout of NET production has been called into question. Cayman's NET Assay Kit offers a DNA-independent readout of NET generation. By employing a specific elastase substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val p-Nitroanilide, this assay measures the enzymatic activity of neutrophil elastase that has been released from NETs through the action of S7 nuclease. Since many NET-associated proteins, including neutrophil elastase, are also released from primary azurophilic granules as soluble mediators that are not associated with NETs, a protocol is included to wash away non-NET associated elastase following NET generation so that only the NET-associated neutrophil elastase remains to be released and assayed. Both PMA and calcium ionophore A-23187 are provided as stimuli for inducing NET generation.

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SPECIFICATIONS

Catalog Number

601010

Form

1 ea

P Type

Assay Kits

Weight

0

Storage Temp

-20

Shipping Temp

-20

Additional Info

Production of neutrophil extracellular traps (NETs) is a key feature of the neutrophil antimicrobial response in which neutrophils weave web-like nets from DNA, histones, and antimicrobial macromolecules upon encountering a bacterial challenge. These NETS, which are also comprised of myeloperoxidase, neutrophil elastase, and lactotransferrin, are extruded to capture and possibly kill any nearby bacteria. Once released, NETs are rapidly cleared by the action of plasma DNAse and by macrophages via scavenger receptor-mediated phagocytosis of the NET/neutrophil complex. Previous methods for detection of NET generation have used fluorescent DNA-intercalating dyes. However, because DNA release can also occur as a result of mechanical cellular injury or necrosis, the use of DNA as a definitive readout of NET production has been called into question. Cayman's NET Assay Kit offers a DNA-independent readout of NET generation. By employing a specific elastase substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val p-Nitroanilide, this assay measures the enzymatic activity of neutrophil elastase that has been released from NETs through the action of S7 nuclease. Since many NET-associated proteins, including neutrophil elastase, are also released from primary azurophilic granules as soluble mediators that are not associated with NETs, a protocol is included to wash away non-NET associated elastase following NET generation so that only the NET-associated neutrophil elastase remains to be released and assayed. Both PMA and calcium ionophore A-23187 are provided as stimuli for inducing NET generation.