Figure 3.

Assessment of NETosis in rmCIRP-treated neutrophils in vitro. A total of 1 × 10⁶ BMDN were treated with the indicated dose of rmCIRP, boiled rmCIRP (5 µg/ml of rmCIRP heated at 80 °C for 5 min), rmCIRP (5 µg/ml) + DNase I (10 U/ml; ThermoFisher Scientific) and phorbol 12-myristate 13-acetate (PMA, 50 nM, Sigma-Aldrich) for 4 h at 37 °C in 5% CO₂. NETs in rmCIRP treated neutrophils were quantified by flow cytometry by staining the cells with anti-mouse Ly6G, MPO, and CitH3 Abs. (A) Representative dot plots of the frequencies of NET forming neutrophils generated from two independent experiments are shown. (B) Bar diagrams representing the quantitative mean values of the frequency of NET forming neutrophils are shown. Data are expressed as means ± SE (n = 4 samples in each group) and compared by using one-way ANOVA and SNK method (*p < 0.05 vs vehicle).