Dextran t 70

After anesthetization with 0.8% pentobarbital sodium, the mice were perfused with pre-cooled saline and the cerebral cortex and hippocampus were immediately removed. The cerebral cortex and hippocampus were weighed and homogenized in cold HBSS (10.0 mM HEPES, 141.0 mM NaCl, 4.0 mM KCl, 1.0 mM MgSO₄•7H₂O, 1.0 mM NaH₂PO₄•2H₂O, 2.5 mM CaCl₂, 10.0 mM glucose and 1.0 mM sodium pyruvate) with Dextran-T70 (32%, Sigma) at a ratio of 1:3:4 (Brain tissues: Buffer: Dextran-T70). The homogenate was centrifuged at 7,450 g at 4 °C for 5 min. The white pellets were separated from the supernatant and homogenized again in 16% Dextran-T70 and centrifuged at 7,450 g at 4 °C for 5 min. The pellets were washed in the same buffer and centrifuged at 3, 500 g, at 4 °C for 5 min. The supernatants were removed and the BMVEC- containing pellet was retained for western blot analysis and ICP-MS.

Bone cement (Palacos) with and without gentamicin was obtained from Heraeus Medical (Hanau, Germany). Gentamicin sulfate, vancomycin, tobramycin, DextranT70 and silver nitrate (all 99% purity) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluoraldehyde-O-pthaldehyde was obtained from Thermo Fisher, Burnaby, BC, Canada. Luria Bertani broth was obtained from Gibco—Thermo Fisher, Burnaby, BC, Canada.

All tissue culture reagents were from Euroclone SpA except ECGS and heparin (Sigma Aldrich). Charcoal, dextran T-70, MTT, trypan blue, methylcellulose (cat #M0512), E2, T3, DHT, sodium acetate, palmitic acid, and free fatty acid-bovine serum albumin (FFA-BSA) were from Sigma Aldrich; rat tail collagen I from Serva; recombinant human VEGF-165 from Peprotech. palmitic acid was used as a conjugate to FFA-BSA. Briefly, a 75 mM stock solution of palmitic acid dissolved in ethanol was diluted 1:10 with a 10% FFA-BSA solution, and incubated overnight at 37°C before every experiment.