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Primary cultures of bovine microvascular retinal endothelial cells (BREC)
are often isolated from freshly isolated calf eyes obtained from a local
abattoir using homogenization and a series of filtration steps. BREC are
often subsequently cultured in endothelial basal medium (EBM, Clonetics,
San Diego, CA) with 10% plasma-derived horse serum (PDHS, Wheaton, Millville,
NJ), 50 mg/l heparin and 50 mg/ml endothelial cell growth factor (ECGF,
Boehringer Mannheim, Chicago) in fibronectin (NYBen Reagents, New York
Blood Center) coated dishes (Costar, Cambridge, MA). Within a week after
initial isolation, BREC can be transferred to new fibronectin-coated dishes
using a cloning ring and Dulbecco's minimal essential medium (DMEM) containing
10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY) and 50 mg/ml
ECGF. The cells most commonly are cultured in 5% CO2 at 37oC and media
changed every three days. Cells are often plated at a density of 2 x 104
cells/cm2 and passaged 1:3 when confluent. Cells at passages 2 to 9 are
usually used for experiments. Endothelial cell homogeneity can be characterized
for their homogeneity by immunoreactivity with anti-factor VIII antibody
and analysis by confocal microscopy. |
