Egm 2 medium bullet kit

Human umbilical vein endothelial cells (HUVECs) were obtained from human umbilical cords [15 (link)], and the cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ using an Endothelial Cell Growth Medium (EGM-2) medium bullet kit (Lonza, Verviers, Belgium). Cells in their third or fourth passage were used for all the reported experiments. Recuperated cells were stained with anti-CD31 FITC-conjugated, anti-CD309 (KDR/VEGFR2) APC-conjugated, and anti-CD146 PE-conjugated antibodies (BD Biosciences, San Jose, California, USA) and evaluated with a MACS Quant Analyzer 10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) flow cytometer; molecular analysis was performed with FlowJo software version 7.6.5 (TreeStar, Inc.).

HPLC-grade acetonitrile and methanol were purchased from Labscan (Dublin, Ireland). Acetic acidof analytical grade (assay >99.5%) was purchased from Fluka (Buchs, Switzerland). Water was purified by using a Milli-Q System (Millipore, Bedford, MA, USA). Other reagents unmarked were of analytical grade. methanol (MeOH) was purchased from Carlo Erba (Rodano, MI, Italy), H₂O₂ was bought from Farmac-Zabban S.p.a. (Calderara di Reno, BO, Italy), NaCO₃, gelatin and Hoechst 33342 were obtained from Sigma-Aldrich (Milan, Italy), poly(vinylidenefluoride-co-trifluoroethylene) [P(VDF-TrFE)] powder (70:30 % mol) was purchased from Piezotech Arkema (Pierre-Benite, France), Methyl ethyl ketone (MEK) was bought from Merck (Darmstadt, Germany). Medium 199 (M199), fetal bovine serum (FBS), penicillin-streptomycin solution, L-glutamine, HEPES buffer were supplied by Hospira S.r.l. (Naples, Italy).
4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolium]-1,3-benzenedisulfonate (WST-1 assay), was purchased from Roche Applied Science (Mannheim, Germany), 5-(and-6)-chloromethyl-2’,7’-dichloro-di-hydro-fluorescein diacetate, acetyl ester (CM-H₂DCFDA) was supplied by Invitrogen. EGM-2 medium bullet kit was bought from Lonza (Verviers, Belgium), alamarBlue^® was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Clonetics, Basel, Switzerland), and cultured according to the manufacturer’s guidelines. The reagents for HUVECs included: EGM-2–medium + bullet kit (Lonza, Clonetics, Italy), HEPES buffered saline solution (Lonza, Clonetics, Italy) and Accutase (Sigma-Aldrich, St. Louis, MO, USA). Cell viability was assessed using WST-1 assay (Takara, Takara Bio Europe, Saint-Germain-en-Laye, France).
Astrocytes were purchased from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). The Gibco Human Astrocytes Kit contains normal human cells and Gibco Astrocyte Medium Kit consisting of base medium (DMEM), N-2 Supplement, and OneShot Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured according to the manufacturer’s guidelines using vessels (75 cm²) coated with Geltrex matrix (Thermo Fisher Scientific, Waltham, MA, USA). Dulbecco’s phosphate-buffered saline (DPBS) with calcium and magnesium ions (Thermo Fisher Scientific, Waltham, MA, USA) was used to rinse culture vessels. The astrocytes were detached from the plate using Accutase (Sigma-Aldrich, St. Louis, MO, USA).