Fig. 5
From: Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain
NCM induced NETosis and NETosed neutrophils induced neuronal cell death. a Schematic diagram of the procedure used to co-culture NCM-treated neutrophils and primary cortical neurons. b HMGB1 levels in NCM of primary cortical neurons (1.6 × 106/4 well) were examined by immunoblotting after 1, 2, or 3 h of NMDA treatment (300 μM, 30 min). c Isolated neutrophils (5 × 105/well) were treated with NCM for 1, 2, 4, 6, 9, and 18 h and CitH3 levels were examined by immunoblotting. (D-E) Neutrophils (5 × 105/well) was pre-incubated with AMD3100 (10 μM), TLR4-IN-C34 (10 μM), PAD inhibitor (10 μM) for 30 min and then treated with NCM or was incubated with NCM, which was pre-incubated with anti-HMGB1 antibody (1 μg/ml), HMGB1 A box (50 ng/ml) for 30 min. CitH3 levels in isolated neutrophils (5 × 105/well) (PMNs) were determined by immunoblotting after 6 h (d) and NET formation was visualized after 6 h of NCM treatment by double immunofluorescent staining using anti-HMGB1 antibody and DAPI (e). f-g NCM-treated PMNs (5 × 105/well) were prepared as it was described in D-E and further included IgG (2 mg/ml) control, and then co-cultured with naïve primary cortical culture (4 × 105/well) for 18 h. Numbers of PI-positive cells were counted (250 μm × 250 μm). Scale bars in E represent 20 μm and those G represent 50 μm. Results are presented as means±SEMs. *p < 0.05, **p < 0.0