Bovine Brain Endothelial Cell Growth Medium

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Cells were transfected 4 hours prior to drug treatments and drug treatments were incubated for 24 h prior to hypoxic exposure in standard cell culture conditions (21% O2) then moved into hypoxic conditions (3% O2) for 24h.	Cell cultures were placed in a special humidified hypoxic chamber for up to 48 h

Upstream tips
Cells were transfected 4 hours prior to drug treatments and drug treatments were incubated for 24 h prior to hypoxic exposure in standard cell culture conditions (21% O2) then moved into hypoxic conditions (3% O2) for 24h.
Protocol tips
Cell cultures were placed in a special humidified hypoxic chamber for up to 48 h

Publication protocol

Bovine Brain microvascular endothelial cells (BMEC) were obtained from Cell Applications INC, (B841-25, San Diego, California) and cultured in endothelial cell basal media (Cell Applications, INC B819-500). For all experiments, BMEC were used from passages 4–8, grown to confluence, and cultured under standard cell culture conditions (21%, ‘normoxia’, 5% CO2) unless otherwise noted.

Cell cultures were placed in a special humidified hypoxic chamber for up to 48 h. Use of an antechamber ensured that once cells became hypoxic, they were never re-exposed to a normoxic environment. The chamber utilized a positive pressure system and was supplied with a gas mixture of 3% O2, 5% CO2, and balance of nitrogen. All relevant permeability tests and preparation for hydrogen peroxide (H2O2) production assays were conducted within the hypoxia chamber to avoid introduction of normoxia during data collection.

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