L glutamine 100x 200 mm

Epstein–Barr Virus (EBV) immortalized PBMCs were provided by Galliera Genetic Bank, from lymphocytes of the two patients, their parents, and four healthy controls, including two children (cell lines 281 and 283) and two adults (cell lines 519 and 550). The immortalized cells were maintained in RPMI 1640 medium without L-Glutamine with phenol red (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS South America origin EU Approved, Euroclone, Milan, Italy), 1% L-Glutamine 100X 200 mM (Euroclone, Milan, Italy), and 1% penicillin–streptomycin solution 100X (Euroclone, Milan, Italy) at 37 °C in a 5% CO₂ atmosphere. Each cell line was synchronized with thymidine treatment, which requires an inoculum of 500,000 cells/mL to be cultured for 24 h in a complete medium enriched with 1% thymidine 200 mM (Merck Life Science, Milan, Italy), capable of blocking cells in the G1 phase, and for the following 24 h in complete medium without thymidine, to restart growth starting from the same stage of the cell cycle, at 37 °C in a 5% CO₂ atmosphere. At the end of the treatment, the cells were washed with HEPES 50 mM (Merck Life Science, Milan, Italy), to prevent cryo-induced pH changes, and pellets of 5 × 10⁶ cells were stored at −80 °C.

RPMI 1640, fetal bovine serum (FBS), penicillin–streptomycin solution 100X, L-glutamine 100X 200 mM, Lympholyte^®-H, and prestained protein SHARPMASS VI MW marker were purchased from Euroclone SpA (Milan, Italy); anti-MMP9 antibody, horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody, protease inhibitor cocktail (100X), and phosphatease inhibitor cocktail (100X) were obtained from Cell Signaling Technology (Danvers, MA, USA); dibutyryl-cAMP, isopropyl β-D-1-thiogalactopyranoside (IPTG), yeast extract, tryptone, PGex6P1, 6,7-Dihydro-7,9-dimethyl-6-(5-methyl-2-furanyl)-11-phenylpyrimido (4′,5′,3,4) pyrrolo (1,2-a) quinoxaline –8,10 (5H,9)-dione (PPQ-102), a reversible and voltage-independent CFTR inhibitor, and the potentiator Ivacaftor (VX770) were purchased from Sigma-Aldrich (Milan, Italy); GSH-sepharose™, ECL Select™ Western Blotting Detection Reagent, and Amersham ™ Protran^® Premium 0.45-μm nitrocellulose were obtained from GE Healthcare (Chicago, IL, USA); Monocytes Isolation Kit II was purchased from Miltenyi Biotec Srl (Bologna, Italy); pEYFP-C1 plasmid was obtained from Clontech Laboratories (Mountain View, CA, USA); QuickChange site-directed mutagenesis kit was from Stratagene (San Diego, CA, USA); BamHI and EcoRI restriction enzymes by Fermentas were purchased from Life Technologies Italia (Monza, Italy).

MET HCL and CXB were generously donated by Dar al Dawa Pharma (Amman, Jordan). Chloroform, diethylamine, triethylamine, as well as HPLC-grade acetonitrile, methanol, and water were purchased from Tedia (Fairfield, OH, USA). A phosphate buffered saline (PBS) tablet, Span^TM 60, and cholesterol were purchased from Sigma Aldrich (Dorset, UK). Ammonium acetate ≥97.0% ACS was purchased from VWR Chemicals BDH^®, glacial acetic acid from Honeywell^® (Raunheim, Germany), Penicillin-Streptomycin Solution 100× from Euroclone^® (Milan, Italy), RPMI 1640 medium with L-Glutamine from Caisson Labs (Smithfield, UT, USA), Dulbecco’s Phosphate Buffered Saline from Euroclone^® (Milan, Italy), L-Glutamine 100X 200 mM from Euroclone^® (Milan, Italy), Sodium Pyruvate Solution from Eurobio^® Scientific (Les Ulis, France), Cytiva^® HyClone Fetal Bovine Serum from Global Life Sciences^® (Pasching, Austria), CellTiter-Glo^® 3D Cell Viability Assay from Promega^® (Mannheim, Germany), Thizolyl Blue Tetrazolium Bromide Powder BioReagent from Sigma-Aldrich Inc.^® (St. Louis, MO, USA), dimethyl sulfoxide (DMSO) from Sigma-Aldrich Inc.^® (St. Louis, MO, USA), and ROS-Glo™ H₂O₂ assay from Promega^® (Mannheim, Germany).