Figure 5

Circulating cfDNA contributes to patient plasma-mediated in vitro NETosis. (a) Neutrophils from healthy volunteers (1 × 10⁶) were stimulated with 20% pooled plasma from healthy volunteers (n = 3), patients with short-CPB (n = 4) and long-CPB (n = 4), respectively. After 3.5 hours, levels of cfDNA/NETs were quantified in culture supernatants. Data are representative for eight independent experiments. **p < 0.01. (b) Circulating cfDNA was isolated from plasma of healthy controls or patients with short (short-CPB) or long (long-CPB) CPB duration, respectively. DNA samples of 4–5 individuals from each group were pooled. Neutrophils from healthy donors (1 × 10⁶) were stimulated with 200 ng DNA for 3.5 hours and cfDNA/NETs were quantified in the culture supernatants (n = 8). *p < 0.05. (c) Pooled plasma from patients with short-CPB (n = 4) and long-CPB (n = 4), respectively, was pre-incubated with DNase I (190 U/ml) for 2 hours at 37 °C. DNase I activity was further inhibited by addition of 5 mM EDTA and neutrophils were incubated in the presence of 20% plasma with or without DNase I pretreatment for 3.5 hours. cfDNA/NETs were quantified in the culture supernatants. Fold change vs. control cells cultured in FCS supplemented medium is depicted. Results are representative for four independent experiments *p < 0.05, **p < 0.01.