Figure 2
NBD-UBIdend demonstrates selectivity for bacteria and A. fumigatus over human cells. (A–C) Co-culture of (A) freshly isolated human neutrophils, (B) freshly isolated human mononuclear cells and (C) human alveolar macrophages retrieved from bronchoalveolar lavage with P. aeruginosa imaged with NBD-UBIdend 5 µM. Plot profiles quantify fluorescence across the dashed line in the NBD channel and counterstain (Syto-82) channel. Each panel shows representative images of at least three independent experiments for each cell type, scale bars represent 10 µm. (D) Quantification of benchtop confocal data demonstrating significantly lower fluorescence on neutrophils, mononuclear cells and BALF macrophages compared to bacteria. Bars show mean fluorescence (+/− SEM) from three independent experiments, where at least three fields of view were assessed. Analysis by Students t-test, *p < 0.05, **p < 0.01. (E,F) 3D reconstruction of benchtop confocal microscope imaging of (E) human alveolar tissue, incubated with P. aeruginosa and labelled with NBD-UBIdend 5 µM and (F) human alveolar tissue, incubated with A. fumigatus and labelled with NBD-UBIdend 10 µM. White arrows indicate specific labelling of bacteria or germinating fungal hyphae, whereas lung epithelial cells are not labelled (blue arrows). Yellow arrows indicate elastin autofluorescence present in the human lung. Images are representative of three independent experiments. Scale bars represent 10 µm.
