Figure 3.
(A) Comparison of the fluorescence spectra of probe (8) (5 µM) (dashed line) after treatment with PMN lysate (solid line). (B) Incubation of PMN lysate (2.5 × 105 cells/ml) leads to an increase in probe fluorescence that is inhibited by the presence of Sivelestat (100 µM) or by the endogenous HNE inhibitor αPI (200 µg/ml). (C) The time-course of probe de-quenching following incubation with purified HNE. Incubation with HNE leads to a rapid increase in fluorescence that is inhibited by the HNE specific inhibitor, Sivelestat (100 µM). X-axis refers to time on plate reader, several seconds after addition of Probe (8), by which time significant de-quenching has occurred. (D) Analysis of the fluorescence increase after 10 minutes with (black columns) or without (white columns) Sivelestat (100 µM) at a range of concentrations of HNE. Statistical analysis by one-way ANOVA with Bonferroni’s post-hoc correction, ****P < 0.0001.