Figure 3
NBD-UBIdend has greater affinity and demonstrates improved signal-to-noise when labelling bacteria in pulmonary surfactant than the linear NBD-UBI and shows stability in ARDS BALF. (A) Quantification of retained fluorescence following a wash and re-imaging on a benchtop confocal microscope. Each bacterial strain and compound were normalised to their pre-wash fluorescence intensities (bacteria were incubated with NBD-UBI at 15 μM or equimolar concentration of NBD-UBIdend at 5 μM). Bars represent means (+/− SEM) of independent experiments where at least three fields of view were assessed, and analysed by a Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. (B) Representative images of P. aeruginosa imaged in the presence of synthetic pulmonary surfactant. Green panels show either NBD-UBI 10 μM or NBD-UBIdend 3.3 μM with red panels showing Syto82 counterstaining, purple panels showing synthetic surfactant and phase contrast images demonstrating surfactant vesicles. (C) Fluorescence quantification of P. aeruginosa imaged with NBD-UBI at 10 μM or NBD-UBIdend at 3.3 μM in the presence of fluorescently labelled high-density surfactant vesicles. Data represents the mean fluorescence of the NBD channel on bacteria compared to surfactant and show a significantly higher bacteria:surfactant fluorescence intensity for NBD-UBIdend than NBD-UBI. Bars represent means (+/− SEM) of three independent experiments where at least three fields of view were assessed, analyses by Student’s t-test, **p < 0.01. (D) Stability of compounds in saline and ARDS BALF demonstrating breakdown of NBD-UBI in ARDS BALF when analysed by MALDI-TOF MS (left panel) the presence of NBD-UBIdend in ARDS BALF when assessed by FTMS (right panel). (E) Representative flow histograms for bacteria when incubated with NBD-UBI (grey histograms) at 15 μM or NBD-UBIdend 5 μM (dotted line) demonstrating a greater fluorescence intensity at eqimolar dye concentrations. (F) Quantification of flow cytometry data for the monomeric form (white bars, normalised) and dendrimeric forms (grey bars) demonstrating between a 2–10 fold increase in fluorescence at eqimolar dye equivalents. Bars represent means (+/− SEM) from three independent experiments, analysis is by students t-test, **p < 0.01, ***p < 0.001.
