Figure 2.

Substrate elasticity affects NETosis in a stimulant-dependent manner. (A) Human neutrophils were seeded on PAA gels coated with collagen-I or fibrinogen. They were stimulated with either PMA (5 nM) or LPS (75 μg/mL), as indicated, and incubated for 3 h. The fluorescence images show the nuclei or extracellular chromatin (Hoechst DNA/chromatin stain) of fixed cells. The images reveal that NETosis depends on all three parameters (substrate elasticity, stimulant, surface coating). See quantification in Figure 3 and images of all conditions in Supplementary Figures S1, S2. (B,C) Neutrophils were seeded on glass coverslips and stimulated with PMA (B) or LPS (C) as described above. After fixation with PFA, cells were stained with anti-MPO antibody (red) and Hoechst against chromatin (blue) and imaged by confocal microscopy to verify NET production. Representative images of neutrophils are shown.