Fig 1: Cytotoxicity of extracellular histones. (a) Time course of Sytox Green neutrophil viability assays with increasing concentrations of mixed histones. Neutrophils were seeded in 96 well plates and allowed to adhere before media was removed and replaced with media containing the indicated concentration of mixed histones and Sytox Green. Fluorescence was measured every hour in a plate reader. The fluorescent signal is proportional to cell death, error bars show 95% confidence intervals (CI) for the mean, n = 3. (b) Mixed histones were fractionated using heparin-Sepharose chromatography to separate core histones (H2, H3 and H4) from H1 and analysed by SDS-PAGE and Coomassie staining. (c) Time course for mixed and fractionated histones (60 µg/ml) incubated with neutrophils in a Sytox Green cell viability assay. (d) Traces from live cell microscopy experiments that included Fluo-4 to follow intracellular Ca2+, propidium iodide for extracellular DNA and histones labelled with Alexa-Fluor 647. Neutrophils were plated onto a glass-bottomed dish in media containing Syto41 and propidium iodide before addition of labelled histones. An early rise in intracellular Ca2+ was followed by increased fluorescence signal for propidium iodide and histones as they bound to exposed DNA. (e–g) Images from a live cell microscopy time lapse video of mixed labelled histones incubated with neutrophils. Dyes used were Syto41 for intracellular DNA (blue), 20 µg/ml Alexa-Fluor 647 labelled histones (red) and propidium iodide for exposed DNA or DNA in cells with compromised membranes (yellow). (e) 20 min incubation showing the lobular structure of neutrophil nuclei. (f) 3 h incubation showing cell surface histone binding and many dead cells (propidium iodide positive). (g) The same image as (f) without the propidium iodide channel to highlight histone staining in red. Cell ‘a’ had histones bound to the cell surface and damage has progressed to the stage where propidium iodide accessed and stained intracellular DNA (yellow in (f)). Labelled histones appear within cell ‘b’, indicating cell membrane damage sufficient to allow histones to enter. Structure ‘c’ is expelled DNA with bound histones, following cell membrane disintegration. The scale bar is 25 µm. Representative images from 1 of n = 3 independent experiments. (h–j). Neutrophils were incubated with histones for 3 h before addition of FITC-labelled anti-myeloperoxidase (MPO) antibody and propidium iodide to visualise DNA. (h) and (i) are individual channels for MPO and DNA, respectively. (j) is the merged image of (h) and (i). (k) Untreated neutrophils stained with Syto41 (DNA) for comparison. Images show expanded/decondensed DNA in the histone treated cells with externalised MPO. Scale = 20 µm. Representative images of n > 50 cells in at least 5 fields of view/sample. (l) Flow cytometric quantitation of MPO externalisation in histone treated (120 µg/ml) neutrophils shows high levels of MPO release similar to that previously observed with ionomycin treatment62. Anti-MPO FITC (green) is compared with isotype control (grey).