Conditioned neutrophil stimulation and antibacterial functions are similar to blood circulating neutrophils. (A) Neutrophil cell-surface markers were quantified on circulating and conditioned neutrophils, either naïve or infected by S. flexneri (MOI 20, 30 minutes). The following cell-surface markers were quantified by flow cytometry: CD35 (C3bR), CD63 (primary granule), CD66b (secondary granules), CD54 (ICAM-1), CD196 (CCR6), and CD62L (l-selectin). Results are expressed as a mean of fluorescence intensity (MFI) calculated on the whole neutrophil population. “ns” indicates P > .05; *P < .05; ***P < .001 (n = 5). (B) S. flexneri phagocytosis and killing efficiencies were assessed with freshly purified or conditioned (20 hours) neutrophils, at MOI 20 during respectively 30 minutes (phagocytosis) and 180 minutes (killing) at 37°C. Intracellular bacteria were counted by plating after gentamycin treatment. Error bars indicate SD. “ns” indicates P > .05 (n = 3). (C) PMA-induced ROS production was assessed by chemoluminescence changes (cpm) on freshly purified and conditioned neutrophils incubated with luminol (10 μM) and stimulated with PMA (200 ng/ml) for 5 minutes. “ns” indicates P > .05 (n = 5). (D) Freshly purified and conditioned neutrophil chemotaxis properties were assessed using Transwells. Neutrophils (2 × 105) were loaded in the upper chamber, and IL-8 (20 nM) was added in the lower buffer. Migrated neutrophils were counted in the lower chamber after 1 hour incubation at 37°C. “ns” indicates P > .05 (n = 5).
