Primary C57BL/6 mouse brain microvascular endothelial cells (MBECs; C57-6023,
Cell Biologics, Chicago, IL) were seeded in 12-well plates coated with
Gelatin-Based Coating Solution (6950, Cell Biologics) for cell assays and
allowed to grow to 80–90% confluency before treatment. Cells were grown in
Complete Mouse Endothelial Cell Medium with supplemental kit (M1168 + kit, Cell
Biologics), at 37°C in 5% CO2. All experiments were completed on cell
passage 5.
Human cerebral microvascular endothelial cells were isolated from post-mortem
brains of donors and transfected with plasmid containing SV40 large T antigen
(HBEC-5i; CRL-3245TM, ATCC®, Manassas, VA). Cells were grown in F12:
DMEM plus 10% FBS and Endothelial Cell Growth Supplement (356,006, Corning) on
0.1% Gelatin (PCS-999–027, ATCC®). To exclude the effect of growth factors,
culture medium was changed to F12:DMEM without serum 4 hours prior to treatment.
All experiments were completed on cell passage 4.
All insulin stimulation and inhibitor treatments were performed at 37°C.
Hyperinsulinemic conditioning was induced in MBECs by incubating cells for
12 hours in 20 nM human recombinant insulin (I9278, Sigma, St. Louis, MO), and
HBEC-5is in 5 nM human recombinant insulin for 12 hours. Time and concentrations
for hyperinsulinemic conditioning were determined in pilot studies, data not
presented here.
Cell Biologics, Chicago, IL) were seeded in 12-well plates coated with
Gelatin-Based Coating Solution (6950, Cell Biologics) for cell assays and
allowed to grow to 80–90% confluency before treatment. Cells were grown in
Complete Mouse Endothelial Cell Medium with supplemental kit (M1168 + kit, Cell
Biologics), at 37°C in 5% CO2. All experiments were completed on cell
passage 5.
Human cerebral microvascular endothelial cells were isolated from post-mortem
brains of donors and transfected with plasmid containing SV40 large T antigen
(HBEC-5i; CRL-3245TM, ATCC®, Manassas, VA). Cells were grown in F12:
DMEM plus 10% FBS and Endothelial Cell Growth Supplement (356,006, Corning) on
0.1% Gelatin (PCS-999–027, ATCC®). To exclude the effect of growth factors,
culture medium was changed to F12:DMEM without serum 4 hours prior to treatment.
All experiments were completed on cell passage 4.
All insulin stimulation and inhibitor treatments were performed at 37°C.
Hyperinsulinemic conditioning was induced in MBECs by incubating cells for
12 hours in 20 nM human recombinant insulin (I9278, Sigma, St. Louis, MO), and
HBEC-5is in 5 nM human recombinant insulin for 12 hours. Time and concentrations
for hyperinsulinemic conditioning were determined in pilot studies, data not
presented here.