N methoxysuccinyl ala ala pro val 7 amido 4 methylcoumarin

Neutrophils (8 × 10⁶) were incubated with live or paraformaldehyde-fixed promastigotes of L. amazonensis in a 1:0.1 neutrophil:parasite ratio or LPS (Escherichia coli O55:B5) 100 ng/mL at 35°C in 5% CO₂. After 3 h incubation, supernatant was collected and aliquots were kept at −80°C until use. The quantification of NETs were performed with the Picogreen dsDNA kit (Invitrogen, Life Technologies), as already described (15 (link)). The NETs-enriched supernatants were treated with DNase (10 U/mL; Fermentas Life Science) or with the elastase inhibitor MeOSuc-AAPV-cmk (10 µg/mL, Calbiochem) for 30 min and then added to monocytes. In some cases, NETs were filtered through a 0.22 µm pore filter to remove the NET scaffold.
The quantification of the elastase in the supernatants was performed as described (18 (link)). Briefly, 50 µL of NETs-enriched supernatants were incubated with the fluorogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (0.25 mM; Sigma-Aldrich). After 1 h at 37°C, the fluorescence was measured in a SpectraMax Paradigm reader (Molecular Devices) at 365–450 nm. A standard curve with recombinant elastase was used to determine the concentration of elastase in the NETs-enriched supernatants.

Neutrophil elastase (NE) activity was measured as described in (36 (link)). Briefly, 50 μL of medium supernatant was collected after 24 h culturing of neutrophils in the presence or absence of PP13 (3 μg/ml), then incubated with the elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (0.25 mM, Sigma) in PBS for 30 min at 37°C, 5% CO₂ in the dark. The reaction product was analyzed at 360/455 nm.

HNE concentration was measured in 50 µl of supernatant using the fluorogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin (Sigma) at a final concentration of 0.25 mM (100 µl final reaction volume). The assay buffer was 50 mM HEPES buffer, pH 7.4, 100 mM NaCl, 0.01% Triton X-100. After 1 h incubation at 37°C, the substrate hydrolysis was measured in a Spectramax Gemini XPS fluorescence microplate reader (Molecular Devices, Menlo Park, CA) with 365/450 nm excitation/emission wavelengths. HNE concentration was determined by using a standard curve of serial dilutions of purified HNE (Elastin Products Company, Inc., Owensville, MO).