Bovine brain extract

Acetylcholinesterase (AChE) from electric eel (Type VI-S, EC 3.1.17.), acetylthiocholine iodide (ATChI), bovine brain extract (Folch type VII), 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid (ferrozine), 2,2-diphenyl-1-picrylhydrazyl (DPPH^•), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), galantamine hydrobromide, sulfanilamide, and rosmarinic acid (96%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Aluminium chloride, ethylenediaminetetraacetic acid (EDTA), and hexamethylenetetramine were purchased from Kemika (Zagreb, Croatia). Butylhydroxytoluene (BHT, ≥99%), luteolin (≥97%), N-(1-naphthyl)ethylenediamine, sodium nitroprusside, and trolox (≥98%) were purchased from Fluka (Buchs, Switzerland). Folin-Ciocalteu's phenol reagent and potassium ferricyanide were obtained from Merck (Darmstadt, Germany). All other chemicals and solvents used were of analytical grade.

MCF10A (CRL-10317), 293T (CRL-11268), A431 (CRL-1555) and HeLa (CCL-2) cell lines
were obtained from ATCC resource center ( http://www.atcc.org). All experiments were performed on cell
lines passaged for <3 months after thawing. 293T, A431 and HeLa cells
were cultured in Dulbecco’s Modified Eagle’s Medium,
DMEM, (Sigma-Aldrich, St Louis, MO, USA). The culture medium was supplemented
with 10% fetal bovine serum (Gibco, Life Technologies, Rockville, MD, USA),
200 U/ml of penicillin and 200 μg/ml
streptomycin (Sigma-Aldrich, St Louis, MO, USA). MCF10A cells were cultured as
described45 (link). Human EC were isolated from umbilical cord
veins, characterized and grown in M199 (Sigma-Aldrich, St Louis, MO, USA)
containing 20% fetal bovine serum (Gibco, Life Technologies, Rockville, MD,
USA), bovine brain extract, heparin (50 μg/ml,
Sigma-Aldrich, St Louis, MO, USA) and penicillin-streptomycin
(200 U/ml, Sigma-Aldrich, St Louis, MO, USA) on gelatin-coated
tissue culture dishes, as previously described46 (link). Human umbilical
cords were kindly donated by O.I.R.M- S. Anna Hospital (Agreement n.619,
19/06/07) with prior written informed consent.

High glucose (4.5 g/L) Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum ((FBS, PAN Biotech, Aidenbach, Germany), L-glutamine, trypsin (2.5% solution of 1:250 trypsin), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), LDH (lactate dehydrogenase), EDTA (ethylenediamimetetraacetic acid), CdCl₂, HgCl₂, or PbCl₂, thiobarbituric acid (TBA), phosphate-buffered saline (PBS), bovine brain extract, FeCl₃, ascorbic acid, butylated hydroxytoluene (BHT) and propyl gallate were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). Quercetin was bought from Extrasynthese (Genay Cedex, France), and cyanidin and cyanidin-3-O-glucoside were bought from Phytolab GmbH & Co.KG (Vestenbergsgreuth, Germany). All solvents used for extraction and chemical analyses were of high purity and were obtained from VWR International s.r.l. (Milan, Italy).