Fig 1: Exosomal release of IL-1a by neutrophils(A) Representative confocal images of peritoneal neutrophils stimulated with LPS or curdlan for 6 h.(B) Quantification of IL-1a and CD63 co-localization using ImageJ (each data point represents a single cell).(C) NTA of EV size distribution and concentration.(D–G) Neutrophils were stimulated in the presence of exosome inhibitor GW4869, and IL-1a and IL-1ß were quantified by ELISA in isolated EVs following lysis (D and F) and in total cell-free supernatants (E and G).(H) Inhibition of EV secretion shown by NTA.(I) Bioactive IL-1 signaling through IL-1R1 reporter cells was measured in isolated exosomes in the absence of detergent lysis (n = 4). Neutralizing antibodies (Abs) to IL-1a, IL-1ß, or both cytokines were included in the reporter assay, and bioactive cytokine concentration was calculated based on a standard curve using recombinant IL-1a and IL-1ß.Two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Experiments in (A) and (B) were repeated three times; (C)–(G) are biological replicates from repeat experiments.