Pathophysiological stimulation with S. aureus (ATCC, 13709) was precedented by previous work35 (link). Isolated neutrophils were allowed to adhere to 96-well culture plates (Costar) in RPMI (2.5×104 cells/well) for 20 min at 37 °C, 5% CO2. Neutrophils were pre-incubated for 30 min with PAD4 inhibitors or vehicle controls (0.1% DMSO final concentration). S. aureus from colonies grown on agar, was shaken in LB at 37 °C for 20 h. The bacteria were washed in ice-cold PBS prior to addition to the neutrophils at 5× MOI (5 bacteria per neutrophil). Following stimulation for 3 h at 37 °C, cells were fixed with 2% PFA. Cells were stained with Hoechst 33342. Plates were imaged on an Opera confocal imaging system (Perkin Elmer) and cells were classified as being 1) lobulated, 2) delobulated or 3) diffused NETS via an algorithm developed using the Acapella software (Perkin Elmer). Images were captured and analysed from 60 fields across 3 wells for each treatment.
Viability was assessed using Cell Titer Glo (Promega) following incubation of human (and mouse) neutrophils with compound alone for identical durations as used in parallel NET studies.
Viability was assessed using Cell Titer Glo (Promega) following incubation of human (and mouse) neutrophils with compound alone for identical durations as used in parallel NET studies.
