Figure 3.

Different strains of Staphylococcus aureus induce NETs (neutrophil extracellular traps), whereas a less virulent coagulase-negative Staphylococcus strain does not induce NETosis (the process of NET formation). A–D, In an in vitro NET release assay, isolated neutrophils (75 000 cells per well) from healthy volunteers were incubated with various bacterial strains (multiplicity of infection [MOI] 100) for 3 hours, and formed NETs were digested to smaller fragments with AluI (4 U) and measured in cell culture supernatants for H3Cit (citrullinated histone H3). A, C, H3Cit absorbances relative to nonstimulated (vehicle-treated) of digested (AluI, red) compared to nondigested (vehicle, blue) samples in the absence (n=8, A) or presence of platelets (6×10⁸ cells/mL, n=5, C). B and D, H3Cit absorbances relative to vehicle of AluI digested samples with corresponding median (interquartile range) in the absence (n=8, B) or presence of platelets (n=5, D). Each single dot represents a single donor. Significance was determined by Kruskal-Wallis test with Dunn post test (B, D). E–H, Overview scanning electron microscopy (SEM) images (secondary electron signal) of NETs formed in the in vitro NET release assay. Scale bar equals 10 μm. I–L, Detailed secondary electron images of NETs incubated with a rabbit anti-H3Cit and a gold-conjugated anti-rabbit antibody. Gold particles were identified on corresponding backscatter electron SEM images and are depicted in green. Scale bars represent 500 nm. Due to different degrees of charging, a different threshold for the gold particles was used in K than in I, J, and L. Complete P values for data presented in B and D are available in Table S1 and S2, respectively. Δnuc indicates nucleases.