Finally samples were centrifuged at 300��g (5 min, 4��C) then at 10,000��g (10 min, 4��C) before GGT determinations. Isolation of neutrophils granules on Percoll gradients Neutrophils Ivacaftor granules were separated as described [23]. Isolated neutrophils (2�C5��107 cells/ml) were pressurized in a nitrogen bomb and the samples were collected dropwise. Nuclei and intact cells were separated by centrifugation and the supernatants were stored on ice. A discontinuos Percoll gradient was prepared by stratifying three Percoll solutions with densities of 1.120, 1.090 and 1.050 g/ml. Supernatants were applied on top of the gradients and centrifuged at 37,000��g (30 min, 4��C). Four main bands were thus identified corresponding to (from bottom): ��-band (containing azurophil granules), ��1-band (specific granules), ��2-band (gelatinase granules), and ��-band (secretory vesicles and plasma membranes).
Cytosol was separated on top of upmost band. The five fractions and fractions among them were harvested through a Pasteur pipette and stored at ?20��C. Fractional GGT analysis by high-performance gel-filtration chromatography Determination of GGT fractions was performed as previously described [24], [25] by a FPLC system (AKTA-purified-10, GE-Healthcare). Separation and quantification of GGT fractions was performed by gel-filtration chromatography (Superose 6 10/300, GE Healthcare) followed by post-column injection of the fluorescent substrate gamma-glutamyl-7-amido-4-methylcoumarin. Intensity of the fluorescence signal was expressed in arbitrary fluorescence units (f.u.
) and the area under chromatographic peaks was proportional to GGT activity. Fractional GGT analysis on activated neutrophils supernatants and solubilised sputum samples were both performed after centrifugation at 10,000��g (30 min, 4��C) followed or not by 100,000��g (120 min, 4��C) ultracentrifugation. Cell lines and culture conditions CFTR-mutated IB3-1 cells derived from bronchial epithelium of a CF patient [26] were obtained from Dr. P. Zeitlin (Johns Hopkins University, MD, USA). IB3-1 cells were routinely grown in LHC-8 medium (Gibco) supplemented with 5% (v/v) foetal bovine serum (FBS). Human alveolar basal epithelial A549 cells [27] were grown in DMEM supplemented with 10% (v/v) FBS and 2 mM L-glutamine (L-Gln). Cell lines were cultured at 37��C in a 5%/95% CO2/air atmosphere.
Western blot analysis The extracellular and cytoplasmatic levels of neutrophilic myeloperoxidase (MPO) were evaluated by western blot analysis of the solubilised sputum supernatants and cells lysates, respectively. The sputum cells were directly lysed in sample buffer (40 mM Tris-HCl pH 6.8, 183 mM ��-mercaptoethanol, 1% (w/v) SDS, 5% (v/v) glycerol), heated at 95��C for 5 min and passed through Entinostat a 23 gauge needle to fragment DNA.