The effect of inhibitors on PMN chemotaxis towards chitosan. Freshly isolated polymorphonuclear neutrophils (PMNs) were resuspended in RPMI 1640 supplemented with 0.1% decomplemented fetal bovine serum, pre-stained with 1 μg/ml calcein-AM for 30 minutes at 37°C and incubated with 0.5 μg/ml pertussis toxin for 90 minutes and seeded on a polycarbonate filter above a well containing 50 μg/ml 80% deacetylated (80 M) chitosan. Alternatively, PMNs were incubated with 10-7 mol/l pyrrolidine for the last 10 minutes of the incubation with calcein-AM. Chemotaxis was performed as described in Figure 1. The percentage inhibition of migration corresponds to the fluorescence of PMNs incubated with the inhibitors that migrated toward 80 M chitosan versus fluorescence of PMN incubated in media that migrated toward 80 M chitosan. This figure represents the results of at least three independent experiments. *P = 0.02 and ***P = 0.0001.