BACKGROUND: The goal of seeding prosthetic conduits with endothelial cells (ECs) has focused attention on the role of EC adhesion molecules. Cell preparation techniques may affect adhesion molecule expression and graft seeding. METHODS: Using fluorescent antibody labeling and flow cytometric analysis, this study examined the effectsof monolayer detachment (scraping vs trypsinization), timing of immunolabeling (pre- vs postdetachment), gene transfection (transfected vs nontransfected), and cell selection (antibiotic vs fluorescence sorting) techniques on beta-1 integrin expression in canine microvascular EC (K9MVEC). RESULTS: Cell scraping resulted in a significantly higher beta-1 integrin mean fluorescence intensity than did cell trypsinization (P < 0.05). No difference was observed with immunolabeling prior to versus following monolayer harvesting. Gene transfection had no significant effect on beta-1 integrin expression. No advantage was observed between cell selection methods (P > 0.05). CONCLUSION: This study suggests that the monolayer harvesting technique employed has a significant impact on beta-1 integrin quantification by flow cytometric analysis. Furthermore, microvascular EC expression of beta-1 integrin was not adversely affected by gene transfection. Copyright 2001 Academic Press.
BACKGROUND: The goal of seeding prosthetic conduits with endothelial cells (ECs) has focused attention on the role of EC adhesion molecules. Cell preparation techniques may affect adhesion molecule expression and graft seeding. METHODS: Using fluorescent antibody labeling and flow cytometric analysis, this study examined the effectsof monolayer detachment (scraping vs trypsinization), timing of immunolabeling (pre- vs postdetachment), gene transfection (transfected vs nontransfected), and cell selection (antibiotic vs fluorescence sorting) techniques on beta-1 integrin expression in canine microvascular EC (K9MVEC). RESULTS: Cell scraping resulted in a significantly higher beta-1 integrin mean fluorescence intensity than did cell trypsinization (P < 0.05). No difference was observed with immunolabeling prior to versus following monolayer harvesting. Gene transfection had no significant effect on beta-1 integrin expression. No advantage was observed between cell selection methods (P > 0.05). CONCLUSION: This study suggests that the monolayer harvesting technique employed has a significant impact on beta-1 integrin quantification by flow cytometric analysis. Furthermore, microvascular EC expression of beta-1 integrin was not adversely affected by gene transfection. Copyright 2001 Academic Press.