FIG 5.

LukAB and not NETosis induces neutrophil lysis. (A to E) Biofilms of the indicated USA300 strains were exposed to neutrophils for 1 h. Neutrophils were then labeled with an anti-CD45 antibody and stained with an Alexa Fluor 488 secondary antibody (green). (F) An anti-IgG antibody was used as an isotype control. (G and H) Wild-type biofilms incubated with Cell Tracker Blue-labeled primary human neutrophils (blue) for 1 h, stained with ethidium homodimer-1 (red, dead/dying neutrophils) and an FITC-labeled anti-CD45 antibody (green). Arrows indicate cells that retained staining with Cell Tracker Blue and anti-CD45 antibody but not ethidium homodimer-1. (I) Percent intact neutrophils undergoing NETosis, calculated for the indicated strains by counting the number of neutrophils that stained with anti-CD45⁺ Cell Tracker Blue as a percentage of the total number of cells per field (anti-CD45 plus Cell Tracker Blue plus EthHD-1), after a 1-h incubation with the respective strains, for 12 fields per strain, from 6 independent experimental replicates. Results are averages from six independent experiments. Images were taken at a total magnification of ×600. Digital zoom (×1.2) was applied to images in G and H, using Imaris software version X 6.4.