Neutrophil stimulation

Purified mouse neutrophils (1 × 10⁶) were resuspended in phenol red-free RPMI and either stimulated with GM-CSF (10 ng/ml) or left untreated for 30 min at 37°C at which point the cells were stimulated with either 5 μM CpG ODN 1826 for 1 h, 10 μg/ml of CL097 for 1 h, 1 μM fMLF for 10 min or 100 ng/ml PMA for 30 min at 37°C as described (Ramadass et al., 2016). Where indicated, neutrophils were incubated with the neutrophil exocytosis inhibitor Nexinhib20 (Johnson et al., 2016) for 1 h before the addition of stimuli. Following the treatments, the cells were spun down at 800 g for 10 min, the cell-free supernatant was collected for ELISA analysis and the cells were stained for flow cytometry analysis.