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. 2012 Sep 19;7(9):e45427. doi: 10.1371/journal.pone.0045427

Figure 2. Identification of TF in NETs released by sepsis neutrophils.

Figure 2

(A) Neutrophils from patients with sepsis (Sepsis PMNs) and control neutrophils treated with sepsis serum (Sepsis serum) or microparticle-depleted sepsis serum (MP-depleted Sepsis serum) were treated for 3 h and the localization of TF on NETs was assessed by confocal microscopy (z stack analysis, 0.3 µm per plane). Treatment with bafilomycin A1 (Bafil) inhibited the release of these structures. One representative out of four independent experiments is shown (DNA labeled with DAPI; blue, anti-TF monoclonal antibody; green) (original magnification 600×). Scale bar represents 10 µΜ. (B) TF levels in proteins isolated from NETs released by sepsis neutrophils or control neutrophils treated with sepsis serum, assessed by immunoblotting. The inhibitory effect of 3-MA in NET release and subsequent presence of TF in NETs is shown. (C) 3-MA and bafilomycin A1 did not affect TF expression in cell lysates from sepsis PMNs incubated for 1 h and (D) in control neutrophils treated with sepsis serum for the same period of time. One out of four independent experiments is shown in B–D. (E–F) TF levels in control neutrophils treated with sepsis serum or microparticle-depleted sepsis serum as demonstrated by western blotting (E) and flow cytometry analysis (F–G). One out of three independent experiments is shown in E–F. Data in (G) are presented as mean ± SD. (‡P<.05 compared to control).