Gentamicin

Primary hippocampal cultures from P0-P2 pups of either sex were performed essentially as described previously^{101 (link),102} . Briefly, postnatal day 0–2 pups were decapitated, the brains quickly removed, hippocampi were dissected, sliced manually, and kept on ice in Hank’s Balanced Salt Solution (HBSS, Biological Industries) supplemented with 20 mM HEPES at pH 7.4. Hippocampus pieces were incubated for 20 min at room temperature (RT) in digestion solution consisting of 5 ml HBSS, CaCl₂ 1.5 mM, EDTA 0.5 mM and 100 units of papain (Worthington) activated with cysteine (Sigma-Aldrich). The brain fragments were then triturated gently two times. Cells were seeded at a density of 80,000–100,000 cells per well on 12 mm #1 glass coverslips coated with poly-D-Lysine (Sigma-Aldrich). Initially, cells were plated in plating medium consisting of Neurobasal-A medium supplemented with 2% B27, 2 mM Glutamax I (Thermo-Fisher Scientific), 5% defined FBS (Biological Industries) and 1 μg/ml gentamicin (Biological Industries). After 24 h, the plating medium was replaced with serum-free culture medium that consisted of Neurobasal-A, 2 mM Glutamax I and 2% B27. Cultures were maintained at 37 °C in a 5% CO₂ humidified incubator for 12–15 days prior to staining or imaging.

The glioma cell line CNS-1 (obtained from Mariano S. Viapiano [23 (link)]) was grown in 96 well plates in DMEM containing 10% fetal calf serum; 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (0.1 mg/mL) under humidified atmosphere containing 5% CO₂. Cells were seeded at 1000 cells/well. Twenty-four hours later, the EDTA and vanadium containing conjugates were added to each plate to give concentrations as indicated in the text. Control experiments using non-cancer cells were conducted with primary bovine brain pericytes and CD34+ human endothelial cells (both obtained and characterized at the Artois University, France [24 (link),25 (link),26 (link)]) treated with the HSA-EDTA-VO⁺⁺ conjugate. These cells were seeded at 15,000 cells/well in ECM medium (Sciencell, Carlsbad, CA, USA), which was composed as follows: 5% fetal calf serum (Gibco, Gaithersburg, MD, USA), ECGS supplements, and 50 mg/mL gentamicin (Biological industries, Beit-Haemek, Israel). Cells were treated the day after. Cell viability was measured after 72 h using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described before [27 (link)]. Experiments were repeated at least 3 times in quadruplicate. IC₅₀ values were calculated from the dose response curves using a median-effect plot.

For propagation rate, cells from each treatment were seeded in 96-well cell-culture plates (Corning, Corning, NY) at a density of 10,000 cells/well and cultured in mammary medium composed of DMEM-F12 medium containing 5% FBS, hydrocortisone (0.5 mg/ml, Sigma), insulin (5 mg/ml, Sigma), gentamicin (50 mg/ml, Biological Industries), streptomycin and penicillin (100 mg/ml and 100 U/ml, respectively), hEGF (10 ng/ml, Merck, Darmstadt, Germany), hFGF2 (10 ng/ml, Merck), heparin (4 mg/ml, Merck), cholera toxin (10 ng/ml, Sigma) and B27 (4 ml stock/ml, Invitrogen, Carlsbad, CA) (10) for the indicated period.