Fig. 4.

Neutrophils release NETs within 5 min of inoculation with S. aureus.

A) Representative 3-dimensional time-course image series of uninhibited neutrophils inoculated with GFP-labelled S. aureus. Vital NETosis is observed at < 20 min, followed by increasing incidence of lytic NETosis at later time points (>20 min post-inoculation with S. aureus). Samples were fixed in 4% PFA at the stated time post-inoculation and subjected to non-permeabilizing immunofluorescence staining. While all samples with fixed with DAPI-containing fixative, some representative images do not have intact PMN in frame, and as such, there is no DAPI signal. B) Time-course quantitation of NET formation in neutrophils with annotation for vital versus lytic NETosis. For immunofluorescence: mitochondria (red), S. aureus (green), nuclei (blue), double-stranded DNA (yellow). For TEM, mitochondria are indicated by white arrows. Data are mean ± SEM of n = 4; Friedman test in H p = 0.0435. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)