Figure 6—figure supplement 1

(A) Neutrophil numbers at the tailfin wound at 4 hpi are not affected by the Lyz:H2az2a transgene in the double transgenic Tg(mpx:GFP)i114;Tg(lyz:h2az2a-mCherry,cmcl2:GFP)sh530 (Figure 6—figure supplement 1—source data 1). (B) Example confocal images of the double transgenic Lyz:H2az2a line showing histone labelling of neutrophils at a tailfin wound. (C) Colocalisation of H2B cerulean fluorescence and SYTOX-green stained DNA during in vitro NETosis. Viable unstimulated Tg(mpx:H2Bcerulean-P2A-mKO2CAAX) neutrophils display blue nuclear and red cytoplasmic fluorescence with minimal intracellular SYTOX green DNA staining. NETs released after calcium ionophore (Cal) stimulation show co-localised extracellular H2Bcerulean fluorescence and SYTOX-green stained DNA. In controls, there is a lack of overlap of blue and green signal in some nuclei demonstrating that co-localisation in the NET taken on the same microscopy settings is not an imaging artefact. (D) In vivo stimulation of transgenic H2Bcerulean zebrafish neutrophils following otic vesicle injection of calcium ionophore (Cal) or PBS injection (control). Unstimulated Tg(mpx:H2Bcerulean-P2A-mKO2CAAX) neutrophils display nuclear cerulean fluorescence surrounded by cytoplasmic immunoreactive myeloperoxidase (arrowheads). Following stimulation with Cal regions of dispersed extracellular H2Bcerulean fluorescence overlay with dispersed Myeloperoxidase immunoreactivity (arrowheads).