EGMTM-2 Endothelial Cell Growth Medium-2 BulletKit

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	Supplemented with 10% fetal bovine serum and EGM-2 SingleQuotes

Protocol tips
Supplemented with 10% fetal bovine serum and EGM-2 SingleQuotes

Publication protocol

HUVECs, ECs and RAECs were purchased from Cell Applications (San Diego, CA, USA) and cultured in EBM-2 plus 10% fetal bovine serum and EGM-2 SingleQuotes (Clonetics, San Diego, CA, USA) in a humidified atmosphere of 5% CO2 and 95% air. Mouse pulmonary ECs were isolated from CatK−/− and CatK+/+ mice by a protocol using PECAM-1-coated Dynabeads (Invitrogen Life Technologies, Carlsbad, CA, USA) and then were cultured in EGM-2 (ref. 38). The isolated ECs retain their in vivo characteristics (purity>85%), such as the expression of cell surface marker PECAM-1 and intracellular adhesion molecule-2. After passage by trypsinization, the cells were cultured in serum-free medium overnight and then treated with 20 ng ml−1 VEGF and/or 400 μM CoCl2 containing the following reagents for the indicated times: CA-074Me (10 μM); E64d (10 μM); CatK inhibitor II, CatL inhibitor V, GM6001 and CatG inhibitor I (10 μM); DAPT (1 μM); and L-685,458 (1 μM). The ECs were also cultured under normoxic (5% CO2 and 95% air) and hypoxic (2% O2, 5% CO2 and 93% N2) conditions for testing on the effects of mechanical hypoxia on Notch1 processing.

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Manufacturer protocol

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