It is generally accepted that the lining vascular endothelium is a major inhibitor of coagulation and thrombus formation. Destroying the protective properties of endothelial cells is shown to have a devastating effect...It is generally accepted that the lining vascular endothelium is a major inhibitor of coagulation and thrombus formation. Destroying the protective properties of endothelial cells is shown to have a devastating effect on coronalry atherosclerotic patients. In our investigation on the role of endothelial cells in thrombocyte activation, the preparation of endothelial cell monolayer is the first important step. The endothelial cells used in our study were prepared from human umbilical vein (HUVEC) and utilized in the experiments at passage 4. The cell culture media contained a basal media supplemented with 5% fetal bovine serum, antibiotics, cortisone and human growth factor. The cells were incubated in a Forma (3193) IR sensored, water jacketed, humidified 5% CO_2 incubator and culture media was replaced with fresh media every 48 hours. Endothelial cell confluence was usually achieved within 2 3 days after plating Institute of Physiology, Ruhr University Bochum, Bochum, Germany (Liu B and Pott L) Platelet Research Unit, University of Muenster, Muenster, Germany (Fassnacht U, Filler T and Zhao B) and the monolayers were utilized for experiments within 5 days. Prior to each experiment, the cells were examined for any abnormalities which might indicate non-confluent or atypical HUVEC monolayers. For calcium mobilization experiments, HUVEC were seeded on 6 well culture plates that were coated with gelatin and were maintained in endothelial growth media without heparin or growth factors for at least 24 hours. For the calcium mobilization cytoskeletal experiments, the media was changed and a basal media supplemented with 5% fetal bovine serum and 20 mmol/L Hepes, pH 7.4 was used to insure constant pH. For the thrombocyte adhesion experiments, washed human thrombocytes were adjusted to a concentration of 2×10~3 cells/ml with a medium containing 1 mmol CaCl_2 and added to the culture falsks. During the experiments, the culture plates or culture falsks were kept in the CO_2 incubator at 37℃. The cultured endothelial cell monolayers have been successfully used for physiological and morphological examinations in thrombocyte research.展开更多
文摘It is generally accepted that the lining vascular endothelium is a major inhibitor of coagulation and thrombus formation. Destroying the protective properties of endothelial cells is shown to have a devastating effect on coronalry atherosclerotic patients. In our investigation on the role of endothelial cells in thrombocyte activation, the preparation of endothelial cell monolayer is the first important step. The endothelial cells used in our study were prepared from human umbilical vein (HUVEC) and utilized in the experiments at passage 4. The cell culture media contained a basal media supplemented with 5% fetal bovine serum, antibiotics, cortisone and human growth factor. The cells were incubated in a Forma (3193) IR sensored, water jacketed, humidified 5% CO_2 incubator and culture media was replaced with fresh media every 48 hours. Endothelial cell confluence was usually achieved within 2 3 days after plating Institute of Physiology, Ruhr University Bochum, Bochum, Germany (Liu B and Pott L) Platelet Research Unit, University of Muenster, Muenster, Germany (Fassnacht U, Filler T and Zhao B) and the monolayers were utilized for experiments within 5 days. Prior to each experiment, the cells were examined for any abnormalities which might indicate non-confluent or atypical HUVEC monolayers. For calcium mobilization experiments, HUVEC were seeded on 6 well culture plates that were coated with gelatin and were maintained in endothelial growth media without heparin or growth factors for at least 24 hours. For the calcium mobilization cytoskeletal experiments, the media was changed and a basal media supplemented with 5% fetal bovine serum and 20 mmol/L Hepes, pH 7.4 was used to insure constant pH. For the thrombocyte adhesion experiments, washed human thrombocytes were adjusted to a concentration of 2×10~3 cells/ml with a medium containing 1 mmol CaCl_2 and added to the culture falsks. During the experiments, the culture plates or culture falsks were kept in the CO_2 incubator at 37℃. The cultured endothelial cell monolayers have been successfully used for physiological and morphological examinations in thrombocyte research.
