Rpmi 1640 medium

Heparin anti-coagulated blood was obtained from the 14 CF patients in accordance with the ethical guidelines. Briefly, peripheral blood mononuclear cells were isolated from dextran sedimentation followed by Ficoll-Hypaque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. The upper plasma layer was aspired and the middle phase containing mononuclear cells was collected and washed in RPMI 1640 medium (Aurogene, Roma, Italy). Viability of the cells was determined by Trypan Blue dye extrusion and resulted in ≥98% viable MNCs.

All the chemicals, including Folin-Ciocalteu’s phenol reagent, polyvinylpyrrolidone (PVP), sodium carbonate, tannic acid (98% purity), aluminum chloride hexahydrate (AlCl₃·6H₂O; Ph Eur purity), quercetin (98% purity), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH; 95% purity), 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH; 97% purity), 2,2-azino-bis (3-thylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; 98% purity), trolox (97% purity), ferrozine (97% purity), iron (II) sulfate heptahydrate (FeSO₄·7H₂O; 99% purity), iron (III) chloride (FeCl₃·6H₂O; 97% purity), hydroxylamine hydrochloride (98% purity), rutin (99% purity), bovine serum albumin, glucose, fructose, sodium azide, iron (II) chloride (FeCl₂·4H₂O; 99% purity), tert-butyl hydroperoxide (tBOOH; 80% purity), and methanol were purchased from Merck (Darmstadt, Germany). RPMI 1640 medium and fetal bovine serum were provided by Aurogene (Rome, Italy).

Human myelomonocytic THP-1 cells were cultured in RPMI 1640 medium (Aurogene, Rome, Italy) supplemented with fetal bovine serum (10%; Aurogene), L-glutamine (2 mM; Aurogene), penicillin (100 IU/mL; Aurogene) and streptomycin (100 mg/mL; Aurogene). Cell culture medium was replaced every 2−3 days, and the cultures were maintained at 37 °C and 5% CO₂ in a fully humidified incubator. The day before each experiment, cells were plated in 48-well culture plates (90.000 cells/well) and were differentiated by treatment with PMA (50 nM, 24 h; Sigma-Aldrich). PMA-differentiated THP-1 cells were washed twice with phosphate-buffered saline (PBS) and primed with LPS (10 μg/mL, 4 h; Sigma-Aldrich) in serum-free medium. Cells were then incubated with compounds dissolved in medium containing 0.1% DMSO for 1 h and cell death was triggered with ATP (5 mM, 90 min; Sigma-Aldrich). MCC950 (Sigma-Aldrich batch #45216 and batch #85021 and from Crysdot (product n. CD31002496; OS05876-18070932) was used in the experiments.