Figure 1
From: Activated neutrophil fluorescent imaging technique for human lungs
(A) Chemical structure of the NAP Smartprobe; (B) the absorbance spectra (with pH) and the emission spectra for NAP in phosphate buffered saline at pH 7.4 (5 µM, λex = 430 nm). Cleaved in the presence of elastase, which liberates carboxyfluorescein from the dendrimer backbone, releases internal quenching mechanism and permits fluorescence. (C) Time course of fluorescence dequenching in the presence of purified HNE. Coloured lines represent control conditions (NAP in the absence of HNE, blue line; NAP and 100 nM HNE in the presence of the specific HNE inhibitor Sivelestat (100 µM), red line; NAP and 50 nM or 100 nM proteinase 3, green lines). Mean point estimate (+ SEM) from three independent experiments. Modest cleavage is shown with supraphysiological concentrations of proteinase 3, an alternative neutrophil specific serine protease. (D) Change in fluorescence with pH (freshly made up pH buffer). NAP (5 µM) shows an increase in fluorescence as pH increases (black circles). There is a steepening of the curve around physiologically relevant pH values. At equivalent concentrations of carboxyfluorescein (15 µM) there is the expected increase in fluorescence as H+ concentration falls (grey triangles). Mean point estimate (± SD, n = 1, in triplicate). (Note the concentration of NAP is 5 µM, but it carries 3 copies of carboxyfluorescein).
