Figure 4.

(A) Addition of probe (8) (5 µM) to freshly isolated human PMNs that have been pharmacologically activated by the calcium ionophore A23187 (10 µM) leads to the rapid appearance of a punctate cell-associated green fluorescence signal (via cleavage of AAPV) when excited at 488 nm. The signal of the probe (green arrow) is observed in the intracellular confocal plane of the PMN nucleus (red arrow) labelled by the fluorescent DNA dye Syto-82 (red) and within the cell boundaries (white arrow). Scale bar: 10 µm. Images are representative of 3 independent experiments. (B) Activation of freshly isolated human PMN with A23187 (10 µM) leads to the rapid appearance of a punctate cell-associated fluorescence signal from the probe (8) when imaged by laser-scanning confocal microscopy (shown at t + 10 minutes). Syto 82 labels the PMN nucleus. Pre-treatment (10 mins) with sivelestat (100 µM) inhibits the appearance of this punctate cell-associated fluorescence whereas pre-treatment with αPI (200 µg/ml) does not. PMN were imaged live in the continued presence of probe (8) and inhibitors. Scale bar: 10 µm. Images are representative of 3 independent experiments.