Fig. 2

HMGB1 from PMPs induces NET formation. a-c Neutrophils from healthy individuals were incubated with PRP from AIS patients (n = 10) and healthy controls (n = 10). Treated neutrophils were co-stained with MPO (green) and H3Cit (red) and observed by confocal microscopy. d NET releasing cells in neutrophils incubated with PPR, PFP and MDP. e The flow cytometry gated of HMGB1 + PMPs is showed. f HMGB1 + PMPs in samples from healthy subjects (n = 10) and AIS patients (n = 10) was detected by flow cytometry. Neutrophils were incubated with PMPs in the presence of Box A (the competitive HMGB1 antagonist). The levels of H3Cit in the supernatant from each group was measured by ELISA (g) and neutrophils were stained with MPO (green) and H3Cit (red) analyzed by confocal microscopy (h-l). The inset scale bars in a, b and h-k are 40 μm. Statistics, Mann-Whitney test (c, f) and ordinary ANOVA (d, g, l). Data are presented as the mean ± SD. ***P < 0.001, and ****P < 0.0001. PMN, polymorphonuclear