Effect of harvesting and sorting on β-1 integrin in canine microvascular cells

K. Todd Piercy, Robert L. Donnell, Stacy S. Kirkpatrick, Bethany L. Mundy, Scott Stevens, Michael Freeman, Mitchell Goldman

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 effects of 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 β-1 integrin expression in canine microvascular EC (K9MVEC). Results. Cell scraping resulted in a significantly higher β-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 β-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 β-1 integrin quantification by flow cytometric analysis. Furthermore, microvascular EC expression of β-1 integrin was not adversely affected by gene transfection.

Original languageEnglish (US)
Pages (from-to)211-216
Number of pages6
JournalJournal of Surgical Research
Volume100
Issue number2
DOIs
StatePublished - Jan 1 2001

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Integrins
Canidae
Endothelial Cells
Transfection
Fluorescence
Cell Adhesion Molecules
Genes
Anti-Bacterial Agents
Transplants
Antibodies

All Science Journal Classification (ASJC) codes

  • Surgery

Cite this

Effect of harvesting and sorting on β-1 integrin in canine microvascular cells. / Piercy, K. Todd; Donnell, Robert L.; Kirkpatrick, Stacy S.; Mundy, Bethany L.; Stevens, Scott; Freeman, Michael; Goldman, Mitchell.

In: Journal of Surgical Research, Vol. 100, No. 2, 01.01.2001, p. 211-216.

Research output: Contribution to journalArticle

Piercy, K. Todd ; Donnell, Robert L. ; Kirkpatrick, Stacy S. ; Mundy, Bethany L. ; Stevens, Scott ; Freeman, Michael ; Goldman, Mitchell. / Effect of harvesting and sorting on β-1 integrin in canine microvascular cells. In: Journal of Surgical Research. 2001 ; Vol. 100, No. 2. pp. 211-216.
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AU - Stevens, Scott

AU - Freeman, Michael

AU - Goldman, Mitchell

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N2 - 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 effects of 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 β-1 integrin expression in canine microvascular EC (K9MVEC). Results. Cell scraping resulted in a significantly higher β-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 β-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 β-1 integrin quantification by flow cytometric analysis. Furthermore, microvascular EC expression of β-1 integrin was not adversely affected by gene transfection.

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