Fig. 8

MMP-9-digested CXCL8 exhibited an increased ability to recruit neutrophils in vivo. Intact or MMP-9-digested CXCL8 was injected into the hindbrain of Tg (coro1a: EGFP; lyz: Dsred) zebrafish larvae at 2 dpf. After 90 min, the larvae were fixed and dual-immunolabeled with anti-GFP and anti-RFP antibodies, after which images were acquired by 3D high-resolution light-sheet microscopy under laser illumination. (a–d) Representative images to show the neutrophils (yellow) and macrophages (green) in the hindbrain of (a) blank or (b) PBS-injected control fish, as well as after injection of either (c) intact or (d) MMP-9-digested CXCL8. Scale bar: 200 μm. (e) Bar chart to show the mean ± standard deviation number of neutrophils in the hindbrain of zebrafish following the various treatments described above. The asterisks (***) represent significant differences when comparing the various groups at p < 0.001, and ‘n.s.’ indicates data that were not significantly different, one-way ANOVA with LSD Post-hoc test. (f) A cell suspension from 4 dpf larvae of the Tg (coro1a: EGFP; lyz: Dsred) line was prepared. An in vitro cell migration assay was performed and the numbers of neutrophils (yellow), and macrophages (green) were quantified. Bar chart to show the mean ± standard deviation numbers of neutrophils and macrophages that migrated towards intact or MMP-9-digested CXCL8. The asterisks (**) indicate intact and digested CXCL8 data that were significantly different at p < 0.01, and ‘n.s.’ indicates data that were not significantly different, two-tailed t-test.