PMC:3359311 / 25931-28194 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3359311","sourcedb":"PMC","sourceid":"3359311","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3359311","text":"Cell Culture\nCF (IB3-1 and CuFi-1) and control (S9 and NuLi-1) cells were obtained from the American Type Culture Collection. IB3-1 cells were derived from a patient expressing the ΔF508 and W1282X mutations and CuFi-1 were derived from a ΔF508 homozygous patient. S9 cells are IB3-1 cells that have been transfected with CFTR using an adeno-associated viral vector and NuLi-1 cells were derived from a patient possessing a wild-type CFTR genotype. THP1-XBlue cells stably express a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-κB and AP-1 (Invivogen). Cells were cultured as recommended by their respective suppliers using standard protocols. S9 and IB3-1 cells were cultured in basal LHC-8 (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM sodium pyruvate. NuLi-1/CuFi-1 cells were cultured in BEBM serum-free medium (Lonza) with supplement bullet kit (EGF, hydrocortisone, bovine pituitary extract, transferrin, bovine insulin, triiodothyronine, epinephrine, retinoic acid), 2 mM L-glutamine, and 1 mM sodium pyruvate. PBMCs from CF patients and controls were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (complete RPMI). THP1-XBlue cells were cultured in complete RPMI with the addition of zeocin (100 µg/ml) to select for cells expressing the SEAP NF-κB/AP-1 reporter. Prior to stimulation, bronchial epithelial cell lines were plated in coated [62] 96-well plates (BD Biosciences) at 3×104 cells/well unless indicated, and allowed to adhere overnight. Plates for S9 and IB3-1 stimulations were coated in a mixture of bovine serum albumin (100 µg/ml), fibronectin (10 µg/ml), and bovine collage type I (30 µg/ml) (BD Biosciences). Plates for NuLi-1 and CuFi-1 were coated with collagen type IV (60 µg/ml) (Sigma Aldrich). PBMCs were plated in 96-well plates at a density of 1.5×105 cells/well in 200 µl (7.5×105 cells/mL). THP-1 reporter cells were differentiated into a macrophage-like phenotype using 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich) for 24 hours at a density of 1×105 cells/well in 200 µl (5×105 cells/ml). 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