Effect of harvesting and sorting on beta-1 integrin in canine microvascular cells.
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).


CONCLUSIONS
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.
Piercy
K T
KT
Department of Surgery, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920, USA. kpiercy@mc.utmck.edu
Donnell
R L
RL
Kirkpatrick
S S
SS
Mundy
B L
BL
Stevens
S L
SL
Freeman
M B
MB
Goldman
M H
MH
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D004285:Dogs; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D019012:Integrin beta1 / Q000032:analysis / Q000235:genetics*; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D019012:Integrin beta1