Quantifying Neutrophil Elastase Activity

Enzymatic activity of NE was evaluated using the NE activity assay kit (Cat#600610; Cayman Chemical; Ann Arbor, MI) as previously described (28 (link)). Neutrophil elastase at 18 mU/ml was used to make a standard curve ranging from 0.156 mU/ml to 10 mU/ml. Standards and samples were added to a black, flat-bottom, 96-well plate. Ten μl of substrate solution consisting of Cell-Based Assay DMF and (Z-Ala-Ala-Ala-Ala)2Rh110 was added to each standard and sample, which is selectively cleaved by elastase to yield the highly fluorescent compound R110. Fluorescence was analyzed with an excitation wavelength of 485 nm and an emission wavelength of 525 nm using a microplate fluorimeter (Varioskan Flash, Thermo Scientific). Kinetic fluorescent measurements were taken every 2 minutes for 2 hours at 37°C. Average fluorescence was taken for each well and quantified in mU/ml with the help of the NE standard.