"Lumen digestion" technique for isolation of aortic endothelial cells from heme oxygenase-1 knockout mice
Endothelial cell dysfunction plays a critical role in the pathogenesis of cardiovascular diseases. Gene targeted mutant, knockout. or transgenic mice are widely used in the laboratory investigation of these disorders. We describe a simple and reproducible "lumen digestion" technique to iso...
| Veröffentlicht in: | BioTechniques. - 1991. - 37(2004), 1 vom: 14. Juli, Seite 84-6, 88-9 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2004
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, U.S. Gov't, P.H.S. DNA Primers Heme Oxygenase (Decyclizing) EC 1.14.14.18 Casp3 protein, mouse EC 3.4.22.- Caspase 3 Caspases |
| Zusammenfassung: | Endothelial cell dysfunction plays a critical role in the pathogenesis of cardiovascular diseases. Gene targeted mutant, knockout. or transgenic mice are widely used in the laboratory investigation of these disorders. We describe a simple and reproducible "lumen digestion" technique to isolate aortic endothelial cells from mice that would be useful for researchers in endothelial cell biology. We used wild-type, homozygote, or heterozygote heme oxygenase-1 null mice from which the aorta is isolated and removed under anesthesia. After cauterizing all the branches, both ends of the aorta are cannulated using an Intramedic PE-20 tube. After flushing the aorta with phosphate-buffered saline (PBS), the lumen is repeatedly instilled (five times) with 50 microL 0.25% trypsin in PBS, incubated for 2 min, and flushed with PBS. The outflow is collected in endothelial cell media with 20% fetal bovine serum. After centrifugation, the endothelial cells in the pellet are resuspended in media and plated in a 24-well tissue culture dish. Following culture for 2 to 3 weeks, the cells demonstrate typical cobblestone appearance, stain positive for the endothelial marker CD31, and are capable of low-density lipoprotein uptake. Following challenge with oxidized lipids, heme oxygenase-1 deficient endothelial cells demonstrate increased susceptibility to cell injury |
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| Beschreibung: | Date Completed 29.03.2005 Date Revised 07.06.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |