NE activity assay

NE was determined by measurement of p-nitroanilide resulting from hydrolysis of the neutrophil elastase-specific peptide, N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. Human NE isolated from human sputum leukocyte elastase (SPC; SE562) was used to create the standard, ranging from 1 μM to 1 × 10⁻⁵ μM. 2 × 10⁵ cells per well was used for either conditioned media or whole cell lysates. Conditioned media was made by incubating 2 × 10⁵ cells in 100 μL of PBS for 1 h. The cell lysates were made with 1× cell lysis buffer with 2 × 10⁵ cells. The manufacturer’s protocol was followed.