N methoxysuccinyl ala ala pro val

PMN (1 × 10⁶) were incubated for 3 h at 37°C 5% CO₂ in presence of bacteria (ratio 1:20) or RNA (10 μg/ml) in RPMI medium without phenol red. After stimulation for NETs release, PMN were treated with Micrococcal Nuclease (MNasa 1 U/ml) in the presence of 1 mM CaCl₂ for 30 min at 37°C in order to detach the NETs that were still attached to the PMN cell body. To stop MNase activity EDTA (5 mM) was added. The activity of elastase in cell-free supernatants was determined using the specific substrate N-methoxysuccinyl-ala-ala-pro-val (1 mM, Sigma-Aldrich, MO, USA) by spectrophotometry at 405 and 550 nm at 4 h post-substrate addition. As a positive control, a suspension of PMN treated with 0.5% Triton X-100 was used.

Neutrophil elastase activity was determined in the same dilutions of plasma samples as the DNA quantification. Overnight, 2 μl of the dilutions was incubated with 10 μl of the specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val (Sigma). After incubation, absorbance at 405 nm was measured in a DeNovix DS-11 Series Spectrophotometer/Fluorometer.