16HBE cells were maintained in DMEM/F12 medium (Invitrogen) with

16HBE cells were maintained in DMEM/F12 medium (Invitrogen) with 10% FCS (Invitrogen), pen 100 U/ml/strep 100 μg/ml, 2 mM L-glutamine (Sigma) and 1 Ug/ml de mTOR inhibitor fungizone and 1.5 g/l sodium bicarbonate (Sigma), and were grown until confluent [49]. Establishment and maintenance of human airway epithelial primary culture cells Primary epithelial cells were obtained from human nasal turbinates (HNT) of patients undergoing turbinectomy as previously described [50]. Briefly, HNT were washed in Dulbecco’s modified Eagle medium DMEM/F12 (Invitrogen) and incubated with 2 Foretinib datasheet mg/ml pronase (Protease XIV; Sigma,) in DMEM/F12 supplemented with pen/strep, at 4°C for 16–20 h under slow rotary agitation (80 rpm.). After

washing, aggregates

were discarded and dissociated cells were filtered using a 30-μm pore filter. The cell suspension was then plated for 2 h at 37°C on plastic dishes (Falcon) to eliminate contaminating fibroblasts. After centrifugation, the supernatant containing the epithelial cells was cultivated in a 1:1 mix (vol:vol) of bronchial epithelium medium BEGM (Lonza Ltd): DMEM/F12 supplemented with Clonetics singlequots (5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 0.5 μg/mL epinephrine, 6.5 ng/mL triiodothyronine, 10 μg/mL transferrin, 0.5 ng/mL human epidermal growth factor, 50 μg/mL gentamicin-amphotericinB, 0.13 mg/mL bovine pituitary extract), 50 U/mL of penicillin-streptomycin and 0.5% fungizone. Heat inactivation of the serum In the experiments devoted to the investigation of the role of the heat-labile component of serum in the production of defensins by the human airway epithelium, selleck products heat inactivation of the

serum, the recognised method for serum decomplementation, was performed as described [51]. Briefly, either human autologous serum or heterologous FCS was heated at 56°C for 30 min. After cultivation of the human respiratory cells under the conditions described above, the cells were exposed to A. fumigatus in the medium containing serum that was either heat-inactivated or not. Exposure of the cells to A. fumigatus conidia or hyphal fragments 5 × 106 of A549, 16HBE or primary culture cells were placed in six well plates in 1.5 ml of the corresponding medium described above check details and grown until confluence. Following washing of A549, 16HBE or primary culture cells with PBS, 106 of A. fumigatus conidia per millilitre of medium were added to the cells for 4, 8 or 18 hours. Exposure to HF was carried out by incubation of the cells for 4, 8 or 18 hours with 20 μl of the standard solution (35 mg of dry weight/ml) obtained from 2 × 108 of resting conidium as described above. All A. fumigatus morphotypes were washed an additional four times in endotoxin-free PBS prior to use to eliminate potential endotoxin contamination. After incubation, unbound conidia were removed by washing wells with PBS prior to RNA purification.

Comments are closed.