The Neutrophil Extracellular Trap (NET) Assay Kit provides a simple and fast method for studying the process of NETosis ex vivo. Notably, The NET Assay Kit does not depend upon the DNA component of NETs, as DNA release can occur independently of NETosis. In this kit, primary neutrophils are stimulated to release NETs with either PMA or a calcium ionophore (both included). Unbound neutrophil elastase is washed away following NET generation. Following digest of NET DNA by S7 nuclease, the supernatant containing neutrophil elastase is added to a substrate, which is selectively cleaved by elastase to yield a 4-nitroaniline product that adsorbs light at 405 nm. Enough reagents are provided to test 24 sample conditions for NET production, with analysis in duplicate.
PMA (1 mM) Assay Reagent: 1 vial/50 µl; -20°C NET Assay S7 Nuclease: 1 vial/50 µl; -20°CNET Assay EDTA: 1 vial/1 ml; RTNET Assay Neutrophil Elastase Substrate: 1 vial/250 µl; -20°CHuman Neutrophil Elastase Assay Reagent: 1 vial/50 µl; -20°CA-23187 (25 mM) Assay Reagent: 1 vial/50 µl; -20°CBovine Serum Albumin Assay Reagent: 1 vial/5 g; 4°CCalcium Chloride (1 M) Assay Reagent: 1 vial/1 ml; RT 96-Well Solid Plate (Colorimetric Assay): 1 plate; RT96-Well Cover Sheet: 1 cover; RT