How Neutrophil Extracellular Traps Become Visible

Figure 1

NETs entrapping Streptococcus (Strep.) suis ΔendAsuis ΔssnA. Immunofluorescence microscopy analysis of NETs released by human neutrophils 4 h after infection with the DNase deletion mutant Strep. suis ΔendAsuis ΔssnA in vitro [25]. Neutrophils and bacteria were centrifuged on poly-L-lysine-coated coverslips and the nuclei were stained with Hoechst (blue). Furthermore, the samples were incubated with a mouse monoclonal antibody against DNA/histone 1 (green, arrows) visualizing long extracellular fibres of released NETs and rabbit anti-Strep. suis antibody to label entrapped Strep. suis (red, arrowheads). The secondary staining was performed with goat anti-mouse Alexa 488-conjugated antibody and goat anti-rabbit Alexa 633-conjugated antibody. The coverslip was embedded in Prolong® Gold antifade. Samples were recorded using a Leica TCS SP5 confocal inverted-base fluorescence microscope with a HCX PL APO 40x 0.75–1.25 oil immersion objective. Settings were adjusted with control preparations using a respective isotype control antibody.