2.2 Neutrophil isolation and incubation
Neutrophils were isolated from citrated whole blood by standard procedures routinely used in our laboratory (Evangelista et al., 2007). In order to mimic CFTR dysfunction, normal neutrophils were treated with CFTRinh-172 (10 µMol/L) for 15 min before experimental use. For adhesion, DMSO or CFTRinh-172-treated neutrophils (4 x 106/ml), resuspended in HEPES-Tyrode buffer (pH 7.4) containing: 129 mmol/L NaC1, 9.9 mmol/L NaHCO3, 2.8 mmol/L KCI, 0.8 mmol/L KH2PO4, 0.8 mmol/L MgCl-6H2O, 5.6 mmol/L Dextrose, 10 mmol/L HEPES, and 1 mmol/L CaC12, were seeded on fibrinogen-coated (200 µg/ml; 200 µl/well for 24 hours at 4°C) 12 well plates and allowed to adhere at 37°C, 5% CO2, in the absence or presence of bacterial endotoxin from Escherichia Coli, serotype 055:B5 (Sigma-Aldrich, Milan, Italy) (10 µg/ml) for 18 h (Totani et al., 2016). In initial experiments, in order to set-up the model, neutrophils were also stimulated by the classical chemoattractants fMLP (1 µMoles/L) and C5a (1 µMoles/L) or Phorbol 12-Myristate 13-Acetate (PMA) (100 nMoles/L). Where indicated, roflumilast-N-oxide (RNO) or vehicle (DMSO) were added to cells 2 min before seeding.